Peptidomimetic macrocycles and uses thereof

ABSTRACT

Methods for treating liquid cancer, determined to lack a p53 deactivation mutation, in a subject are provided. Also provided are peptidomimetic macrocycles for use in treatment of a liquid cancer, determined to lack a p53 deactivation mutation, in a subject.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No. 15/074,794, filed Mar. 18, 2016, which claims the benefit of U.S. Provisional Patent Application No. 62/136,357, filed on Mar. 20, 2015, and U.S. Provisional Patent Application No. 62/232,275, filed on Sep. 24, 2015, the entirety of each of which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 14, 2018, is named 35224-806_301_SL.txt and is 1,199,070 bytes in size.

BACKGROUND OF THE INVENTION

The human transcription factor protein p53 induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase MDM2 (also known as HDM2 or human double minute 2) negatively regulates p53 function through a direct binding interaction that neutralizes the p53 transactivation activity, leads to export from the nucleus of p53 protein, and targets p53 for degradation via the ubiquitylation-proteasomal pathway. Loss of p53 activity, either by deletion, mutation, or MDM2 overexpression, is the most common defect in human cancers. Tumors that express wild type p53 are vulnerable to pharmacologic agents that stabilize or increase the concentration of active p53. In this context, inhibition of the activities of MDM2 has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. MDMX (also known as MDM4, HDM4 or human double minute 4) has more recently been identified as a similar negative regulator of p53, and studies have revealed significant structural homology between the p53 binding interfaces of MDM2 and MDMX.MDMX has also been observed to be overexpressed in human tumors. The p53-MDM2 and p53-MDMX protein-protein interactions are mediated by the same 15-residue alpha-helical transactivation domain of p53, which inserts into hydrophobic clefts on the surface of MDM2 and MDMX. Three residues within this domain of WT p53 (F19, W23, and L26) are essential for binding to MDM2 and MDMX.

Provided herein are compounds capable of binding to and modulating the activity of p53, MDM2 and/or MDMX. Also provided herein are pharmaceutical formulations comprising p53-based peptidomimetic macrocycles that modulate an activity of p53. Also provided herein are pharmaceutical formulations comprising p53-based peptidomimetic macrocycles that inhibit the interactions between p53, MDM2 and/or MDMX proteins. Further, provided herein are methods for treating diseases including but not limited to liquid cancers and other hyperproliferative diseases.

SUMMARY OF THE INVENTION

Described herein are methods of treating a liquid tumor determined to lack a p53 deactivating mutation, in a human subject in need thereof where the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

Further disclosed herein are methods of treating a liquid tumor that lacks a p53 deactivating mutation, in a human subject in need thereof where the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

Further disclosed herein are methods of treating a liquid tumor that has a p53 deactivating mutation in a p53 gene, in a human subject in need thereof where the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

Further disclosed herein are methods of treating a liquid tumor in a human subject in need thereof, where the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, where the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins and where the liquid tumor is not negative for p53 protein expression (such as liquid tumors that express wild-type p53 protein or mutated p53 protein with partial functionality).

Further disclosed herein are methods of treating a liquid tumor in a human subject in need thereof, where the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins and wherein the liquid tumor expresses a p53 protein with a gain of function mutation (such as a super apoptotic p53).

Further disclosed herein are methods of treating a liquid tumor in a human subject in need thereof, wherein the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a therapeutically equivalent amount of a pharmaceutically acceptable salt thereof, where the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins and wherein the liquid tumor express a p53 protein with a mutation that causes a partial loss of function.

Further disclosed herein are methods of treating a liquid tumor a human subject in need thereof, wherein the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins and wherein cells in the liquid tumor express p53 from only a single genomic copy of the p53 gene (for example where the cells have a copy loss mutation, e.g., are haploinsufficient).

Further disclosed herein are methods of treating a liquid tumor a human subject in need thereof wherein the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, where the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins and wherein the liquid tumor express a p53 protein with one or more silent mutations.

Further disclosed herein are methods of treating a liquid tumor a human subject in need thereof wherein the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a therapeutically equivalent amount of a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins and where cells in the liquid tumor are negative for p53 expression.

In some embodiments, the cells in the liquid tumor have the p53 deactivating mutation in one copy of the p53 gene. In another embodiment, the cells in the liquid tumor have a second p53 deactivating mutation in a second copy of a p53 gene. In another embodiment, the p53 deactivating mutation in one copy of the p53 gene is the same as the second p53 deactivating mutation in the second copy of a p53 gene. In another embodiment, the p53 deactivating mutation in one copy of the p53 gene is different from the second p53 deactivating mutation in the second copy of a p53 gene. In another embodiment, the p53 deactivating mutation in the p53 gene results in the lack of p53 protein expression from the p53 gene or in expression of partial a p53 protein with partial loss of function. In another embodiment, the second p53 deactivating mutation in the second copy of a p53 gene results in the lack of p53 protein expression from the p53 gene or in expression of partial a p53 protein with partial loss of function.

In some embodiments, the cells of the liquid tumor have at least one mutation in a copy of a p53 gene, where the mutation eliminates or reduces the activity of a p53 protein expressed from the copy of the p53 gene, as compared to wild type p53 expressed from a copy of a non-mutated p53 gene. In another embodiment, the at least one mutation in a copy of a p53 gene is a non-synonymous mutation. In another embodiment, the at least one mutation in a copy of a p53 gene is a synonymous mutation. In another embodiment, the at least one mutation in a copy of a p53 gene is a synonymous mutation, where the synonymous mutation does not change amino acid sequence of a p53 protein expressed from the copy of the p53 gene. In another embodiment, the at least one mutation in a copy of a p53 gene is a synonymous mutation, where the synonymous mutation increases or decreases binding of a micro-RNA to a mRNA. In another embodiment, the at least one mutation in a copy of a p53 gene is a synonymous mutation, where the synonymous mutation alters (e.g., increases or decreases) the half-life of mRNA.

Further disclosed herein are methods of treating a liquid tumor in a human subject in need thereof where the method comprises administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of a peptidomimetic macrocycle or a therapeutically equivalent amount of a pharmaceutically acceptable salt thereof, where the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

In some embodiments, the peptidomimetic macrocycle disrupts the interaction between p53 and MDM2 and MDMX.

In some embodiments, the method comprises determining the lack of the p53 deactivating mutation in the liquid tumor prior to the administration of the pharmaceutical composition. In another embodiment, determining the lack of the p53 deactivating mutation comprises confirming the presence of wild type p53 in the liquid tumor. In another embodiment, the method comprises determining a presence of a p53 gain of function mutation in the liquid tumor. In another embodiment, the method comprises determining a presence of a deactivating mutation of p53 in the liquid tumor. In another embodiment, the method comprises determining a presence of a copy loss mutation of p53 in the liquid tumor. In another embodiment, the method comprises determining a presence of a partial loss of function mutation of P53 in the liquid tumor. In another embodiment, the method comprises confirming the lack of the p53 deactivating mutation in the liquid tumor prior to the administration of the pharmaceutical composition. In another embodiment, the confirming the lack of the p53 deactivating mutation comprises confirming the presence of wild type p53 in the liquid tumor. In another embodiment, the method comprises confirming a presence of a p53 gain of function mutation in the liquid tumor. In another embodiment, the method comprises confirming a presence of a deactivating mutation of p53 in the liquid tumor. In another embodiment, the method comprises confirming a presence of a copy loss mutation of p53 in the liquid tumor. In another embodiment, the method comprises comprising confirming a presence of a partial loss of function mutation of P53 in the liquid tumor.

In some embodiments, the determining or the confirming is performed within 1-15 months prior to the administration of the pharmaceutical composition. In another embodiment, the determining or the confirming is performed within 1-12 months prior to the administration of the pharmaceutical composition. In another embodiment, the determining or the confirming is performed within 1-3 months prior to the administration of the pharmaceutical composition. In another embodiment, the determining or the confirming is performed within 1 month prior to the administration of the pharmaceutical composition. In another embodiment, the determining or the confirming is performed within 21 days prior to the administration of the pharmaceutical composition. In another embodiment, the determining or the confirming is performed up to about 1 year prior to the administration of the pharmaceutical composition. In another embodiment, the determining or the confirming is performed up to about 2 years prior to the administration of the pharmaceutical composition. In another embodiment, the determining or the confirming is performed up to about 3 years prior to the administration of the pharmaceutical composition.

In some embodiments, the treatment results in re-activation of the p53 pathway, decreased liquid cancer cell proliferation, increased p53 protein, increased p21, and/or increased apoptosis in the human subject.

In some embodiments, the pharmaceutical composition is administered two or three times a week. In another embodiment, the pharmaceutical composition is administered two times a week. In another embodiment, the pharmaceutical composition is administered once every 2 or 3 weeks. In another embodiment, the pharmaceutical composition is administered once every 1 or 2 weeks. In another embodiment, the pharmaceutical composition is administered on days 1, 4, 8, and 11 of a 21-day cycle. In another embodiment, the pharmaceutical composition is administered on days 1, 8, and 15 of a 28-day cycle.

In some embodiments, the amount of the compound administered is about 0.5-30 mg per kilogram body weight of the human subject. In another embodiment, the amount of the compound administered is about 0.5-20 mg per kilogram body weight of the human subject. In another embodiment, the amount of the compound administered is about 0.5-10 mg per kilogram body weight of the human subject. In another embodiment, the amount of the compound administered is about 0.04 mg, about 0.08 mg, about 0.16 mg, about 0.32 mg, about 0.64 mg, about 1.25 mg, about 1.28 mg, about 1.92 mg, about 2.5 mg, about 3.56 mg, about 3.75 mg, about 5.0 mg, about 7.12 mg, about 7.5 mg, about 10 mg, about 14.24 mg, about 15 mg, about 20 mg, or about 30 mg per kilogram body weight of the human subject. In another embodiment, the amount of the compound administered is about 1.92 mg, about 3.75 mg, about 7.5 mg, about 15.0 mg, or about 30.0 mg per kilogram body weight of the human subject and the compound is administered two times a week. In another embodiment, the amount of the compound administered is about 1.28 mg, about 2.56 mg, about 5.0 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered two times a week. In another embodiment, the amount of the compound administered is about 1.92 mg, about 3.75 mg, about 7.5 mg, about 15.0 mg, or about 30.0 mg per kilogram body weight of the human subject and the compound is administered once a week. In another embodiment, the amount of the compound administered is about 1.28 mg, about 2.56 mg, about 5.0 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered once a week. In another embodiment, the amount of the compound administered is about about 1.92 mg, about 3.75 mg, about 7.5 mg, about 15.0 mg, or about 30.0 mg mg per kilogram body weight of the human subject and the compound is administered once a day three, five or seven times in a seven day period. In another embodiment, the compound is administered intravenously once a day, seven times in a seven day period. In another embodiment, the amount of the compound administered is about 1.28 mg, about 2.56 mg, about 5.0 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered once a day three, five or seven times in a seven day period. In another embodiment, the compound is administered intravenously once a day, seven times in a seven day period.

In some embodiments, the compound is administered over a period of 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h. In another embodiment, the compound is administered over a period of 0.25-2 h. In another embodiment, the compound is gradually administered over a period of 1 h. In another embodiment, the compound is gradually administered over a period of 2 h.

In some embodiments, the treatment results in about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction in the number of liquid cancer cells within a period of 1 month after treatment initiation. In another embodiment, the treatment results in at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the number of liquid cancer cells within a period of 1 month after treatment initiation. In another embodiment, the treatment results in about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction in the number of liquid cancer cells within a period of 1 year after treatment initiation. In another embodiment, the treatment results in at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the number of liquid cancer cells within a period of 1 year after treatment initiation. In another embodiment, the treatment results in about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction the number of liquid cancer cells within a period of 6 months after treatment initiation. In another embodiment, the treatment results in at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the number of liquid cancer cells within a period of 6 months after treatment initiation. In another embodiment, the treatment results in about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction in the number of liquid cancer cells within a period of 3 months after treatment initiation. In another embodiment, the treatment results in at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the number of liquid cancer cells within a period of 3 months after treatment initiation.

In some embodiments, the liquid cancer is a stable disease.

In some embodiments, the treatment results in an increased survival time of the human subject as compared to the expected survival time of the human subject if the human subject was not treated with the compound. In another embodiment, the increase in the survival time of the human subject is at least 30 days. In another embodiment, the increase in the survival time of the human subject is at least 3 months. In another embodiment, the increase in the survival time of the human subject is at least 6 months. In another embodiment, the increase in the survival time of the human subject is at least 1 year.

In some embodiments, the in vivo circulating half-life of the compound is about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h or 12 h. In another embodiment, the in vivo circulating half-life of the compound is about 4 h. In another embodiment, in vivo circulating the half-life of the compound is about 6 h.

In some embodiments, the biological tissue half-life of the compound is about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h or 12 h. In another embodiment, the biological tissue half-life of the compound is about 10 h.

In some embodiments, the human subject is refractory and/or intolerant to one or more other treatment of the liquid cancer. In another embodiment, the human subject has had at least one unsuccessful prior treatment and/or therapy of the liquid cancer.

In some embodiments, the liquid cancer expresses wild-type p53 protein.

In some embodiments, the liquid cancer is selected from a group consisting of liquid lymphoma, leukemia, and myeloma. In another embodiment, the liquid cancer is a liquid lymphoma. In another embodiment, the liquid cancer is a leukemia. In another embodiment, the liquid cancer is an acute leukemia. In another embodiment, the acute leukemia is an acute myeloid leukemia (AML). In another embodiment, the acute leukemia is an acute lymphoid leukemia (ALL). In another embodiment, the liquid cancer is a myeloma. In another embodiment, the liquid cancer is not a HPV positive cancer. In another embodiment, the liquid cancer is not HPV positive cervical cancer, HPV positive anal cancer or HPV positive head and neck cancer, such as oropharyngeal cancers.

In some embodiments, the compound is administered intravenously.

In some embodiments, the method further comprises administering in addition to the compound, a therapeutically effective amount of at least one additional therapeutic agent and/or therapeutic procedure to the human subject.

In some embodiments, the human subject exhibits a complete response to the treatment. In another embodiment, the human subject exhibits a partial response to the treatment.

In some embodiments, the liquid cancer is a progressive disease. In another embodiment, the liquid cancer is a stable disease.

In some embodiments, the method further comprises determining clinical activity of the administered compound. In another embodiment, the clinical activity is determined by an imaging method selected from a group consisting of computed tomography (CT), magnetic resonance imaging (MRI), bone scanning, and positron emission tomography (PET) scan. In another embodiment, the PET scan uses one or more tracers. In another embodiment, the one or more tracers comprises ¹⁸F-fluorodeoxyglucose (FDG), ⁶⁴Cu diacetyl-bis(N⁴-methylthiosemicarbazone) (ATSM), ¹⁸F-fluoride, 3′-deoxy-3′-[¹⁸F]fluorothymidine (FLT), ¹⁸F-fluoromisonidazole (FMISO), Gallium, Technetium-99m, or Thallium.

In another embodiment, the method further comprises obtaining a biological sample from the human subject at one or more specific time-points and analyzing the biological sample with an analytical procedure. In another embodiment, the analytical procedure is selected from a group comprising blood chemistry analysis, chromosomal translocation analysis, needle biopsy, tissue biopsy, fluorescence in situ hybridization, laboratory biomarker analysis, immunohistochemistry staining method, flow cytometry, or a combination thereof. In another embodiment, the method comprises tabulating and/or plotting results of the analytical procedure. In another embodiment, the one or more specific time-points comprise a time-point before the administration of the compound to the human subject. In another embodiment, the one or more specific time-points comprise a time-point after the administration of the compound to the human subject. In another embodiment, the one or more specific time-points comprise a time-point before and a time-point after the administration of the compound to the human subject. In another embodiment, the method further comprises comparing the biological samples collected before and after the administration of the compound to the human subject. In another embodiment, the one or more specific time-points comprise multiple time-points before and after the administration of the compound to the human subject. In another embodiment, the method further comprises comparing the biological samples collected at the multiple time-points. In another embodiment, the biological sample is used for biomarker assessment. In another embodiment, the biological sample is used for pharmacokinetic assessment. In another embodiment, the pharmacokinetic assessment comprises studying the level of the peptidomimetic macrocycle and/or its metabolites in the biological sample at the specific time-points. In another embodiment, the biological sample is a blood sample or a bone marrow sample. In another embodiment, the biological sample is used for pharmacodynamic assessment. In another embodiment, the pharmacodynamic assessment comprises studying the level of p53, MDM2, MDMX, p21 and/or caspase in the biological sample at the specific time-points. In another embodiment, the biological sample is a liquid cancer cell specimen. In another embodiment, the biological sample is used for immunogenicity assays.

In some embodiments, the method further comprises selecting and/or identifying at least one circulating tumor cells (CTC) or a mononuclear blood cells (MNBC) in the human subject prior to the administration of the compound to the human subject. In another embodiment, the method further comprises measuring the number of circulating tumor cells (CTCs) or mononuclear blood cells (MNBCs) at one or more specific time-points, where the number of circulating tumor cells (CTCs) or mononuclear blood cells (MNBCs) is the total number of at least one circulating tumor cells (CTC) or a mononuclear blood cells (MNBC) at the specific time-point. In another embodiment, the method further comprises measuring a baseline sum diameter, where the baseline sum diameter is a sum of the diameters of the at least one circulating tumor cells (CTC) or a mononuclear blood cells (MNBC) prior to the administration of the compound to the human subject. In another embodiment, the treatment results in disappearance of the least one circulating tumor cells (CTC) or a mononuclear blood cells (MNBC). In another embodiment, the treatment the number of CTCs and/or MNBCs is reduced. In another embodiment, the one or more specific time-points, comprise a time-point after the treatment. In another embodiment, the number of CTCs and/or MNBCs at the time-point after the treatment is at least 30% less than the baseline number of CTCs and/or MNBCs. In another embodiment, the treatment results in neither sufficient increase nor a sufficient decrease in the number of CTCs and/or MNBCs at the one or more specific time-points, taking as reference the baseline number of CTCs and/or MNBCs.

In some embodiments, the peptidomimetic macrocycle is not an inhibitor of cytochrome P450 isoforms.

In some embodiments, the treatment results in essentially no dose-limiting thrombocytopenia. In another embodiment, the treatment causes essentially no adverse effects in a normal-hematopoietic organ and/or tissue. In another embodiment, the treatment results in essentially no adverse event in the human subject that is possibly, probably, or definitely related to the administration of the compound. In another embodiment, the treatment results in essentially no serious adverse event in the human subject that is probably, probably, or definitely related to the administration of the compound.

In some embodiments, the lack of p53 deactivation mutation in the liquid cancer is determined by DNA sequencing of the nucleic acid encoding the p53 protein. In another embodiment, the lack of p53 deactivation mutation in the liquid cancer is determined by RNA array based testing. In another embodiment, the lack of p53 deactivation mutation in the liquid cancer is determined by RNA analysis. In another embodiment, the lack of p53 deactivation mutation in the liquid cancer is determined by polymerase chain reaction (PCR). In another embodiment, the p53 deactivating mutation comprises mutations in DNA-binding domain of the protein. In another embodiment, the p53 deactivating mutation comprises missense mutation. In another embodiment, the p53 deactivating mutation is a dominant deactivating mutation. In another embodiment, the p53 deactivating mutation comprises one or more mutations selected from a groups consisting of V173L, R175H, G245C, R248W, R249S and R273H. In another embodiment, the p53 deactivating mutation comprises one or more of mutations shown in Table 1.

Also disclosed herein are methods of treating liquid cancer in a human subject determined to lack a p53 deactivating mutation, where the method comprises administering to the human subject 0.5-20 mg of a peptidomimetic macrocycle per kilogram body weight of the human subject or a pharmaceutically acceptable salt thereof on days 1, 8 and 15 of a 28-day cycle.

In some embodiments, the peptidomimetic macrocycle per kilogram body weight of the human subject or a pharmaceutically acceptable salt thereof is administered to the human subject.

In some embodiments, the amount of the peptidomimetic macrocycle entered on day 8 and/or day 15 is greater than the amount of the peptidomimetic macrocycle entered on day 1. In another embodiment, the amount of the peptidomimetic macrocycle entered on day 8 and/or day 15 is equal than the amount of the peptidomimetic macrocycle entered on day 1. In another embodiment, the amount of the peptidomimetic macrocycle entered on day 1 and/or day 8 is greater than the amount of the peptidomimetic macrocycle entered on day 15. In another embodiment, equal amounts of the peptidomimetic macrocycle are administered on days 1, 8 and 15.

In some embodiments, the 28-day cycle is repeated 2 or 3 times.

Also disclosed herein are methods of treating liquid cancer in a human subject determined to lack a p53 deactivating mutation, where the method comprises administering to the human subject 0.25-10 mg of a peptidomimetic macrocycle per kilogram body weight of the human subject or a pharmaceutically acceptable salt thereof on days 1, 4, 8 and 11 of a 21-day cycle.

In some embodiments, 0.25-5 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject or a pharmaceutically acceptable salt thereof is administered to the human subject.

In some embodiments, the amount of the peptidomimetic macrocycle entered on day 4, 8, and/or day 11 is greater than the amount of the peptidomimetic macrocycle entered on day 1. In another embodiment, the amount of the peptidomimetic macrocycle entered on day 4, 8, and/or day 11 is equal than the amount of the peptidomimetic macrocycle entered on day 1. In another embodiment, the amount of the peptidomimetic macrocycle entered on day 1, 4, and/or day 8 is greater than the amount of the peptidomimetic macrocycle entered on day 11. In another embodiment, equal amounts of the peptidomimetic macrocycle is administered on days 1, 4, 8, and 151.

In some embodiments, the 21-day cycle is repeated 2 or 3 times.

In some embodiments, the peptidomimetic macrocycle comprises an amino acid sequence which is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to an amino acid sequence in any of Table 3, Table 3a, Table 3b, and Table 3c, where the peptidomimetic macrocycle has the formula:

wherein: each A, C, and D is independently an amino acid; each B is independently an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-]; each E is independently an amino acid selected from the group consisting of Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine); each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or forms a macrocycle-forming linker L′ connected to the alpha position of one of the D or E amino acids; each R₃ independently is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅; each L and L′ is independently a macrocycle-forming linker of the formula-L₁-L₂-; each L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionally substituted with R₅; each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃; each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue; each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue; each v is independently an integer from 0-1000; each w is independently an integer from 0-1000, for example, 0-500, 0-200, 0-100, 0-50, 0-30, 0-20, 0-10, 0-5, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, 1-10, 1-5, 3-1000, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; u is an integer from 1-10; each x, y and z is independently an integer from 0-10; and each n is independently an integer from 1-5.

In some embodiments, the peptidomimetic macrocycle has formula:

wherein: each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually an amino acid, where at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8) or Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9), where each X₄ and X₁₁ is independently an amino acid; each D is independently an amino acid; each E is independently an amino acid selected from the group consisting of Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine); each R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or forms a macrocycle-forming linker L′ connected to the alpha position of one of the D or E amino acids; each L or L′ is independently a macrocycle-forming linker each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue; each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue; v is an integer from 1-1000; and w is an integer from 0-1000.

In some embodiments, at least one of the macrocycle-forming linker has a formula-L₁-L₂-, where L₁ and L₂ are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionally substituted with R₅; each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃; each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅; and n is an integer from 1-5.

In some embodiments, w is an integer from 0-1000, for example, 0-500, 0-200, 0-100, 0-50, 0-30, 0-20, 0-10, 0-5, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, 1-10, 1-5, 3-1000, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, Xaa₅ is Glu or an amino acid analog thereof. In another embodiment, each E is independently Ala (alanine), Ser (serine) or an analog thereof. In another embodiment, [D]_(v) is-Leu₁-Thr₂.

In some embodiments, w is 3-10. In another embodiment, w is 3-6. In another embodiment, w is 6-10. In another embodiment, w is 6.

In some embodiments, v is 1-10. In another embodiment, v is 2-10. In another embodiment, v is 2-5. In another embodiment, v is 2.

In some embodiments, L₁ and L₂ are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, or heterocycloarylene, each being optionally substituted with R₅. In another embodiment, L₁ and L₂ are independently alkylene or alkenylene. In another embodiment, L is alkylene, alkenylene, or alkynylene. In another embodiment, L is alkylene. In another embodiment, L is C₃-C₁₆ alkylene. In another embodiment, L is C₁₀-C₁₄ alkylene.

In some embodiments, R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. In another embodiment, R₁ and R₂ are H. In another embodiment, R₁ and R₂ are independently alkyl. In another embodiment, R₁ and R₂ are methyl.

In some embodiments, x+y+z=6.

In some embodiments, u is 1.

In some embodiments, the peptidomimetic macrocycle comprises at least one amino acid which is an amino acid analog.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 163):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 124):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 123):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 108):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 397):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 340):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 454):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 360):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 80):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 78):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 16):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 169):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 324):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 258):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 446):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 358):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 464):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 466):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 467):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 376):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 471):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula: (SEQ ID NO: 473)

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 475):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 476):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 481):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 482):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 487):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 572):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 572):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has formula (SEQ ID NO: 1500):

or a pharmaceutically acceptable salt thereof.

Also disclosed herein are methods of identifying one or more liquid cancer biomarkers in a human subject lacking a p53 deactivating mutation, comprising administering to the human subject a therapeutically effective amount of a peptidomimetic macrocycle.

In some embodiments, the biomarkers are p53 status, MDM2 expression level or MDMX expression level.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 shows human wild type P53 coding and protein sequence (SEQ ID NO: 1501).

FIG. 2 shows peptide 1 yielded robust apoptotic responses in p53 wild-type hematopoietic cell lines.

FIG. 3 shows peptide 1 yielded on-mechanism p21 pharmacodynamic responses in p53 wild-type hematopoietic cell lines.

FIG. 4 shows peptide 1 selectively killed p53 wild-type cancer cells in a representative hematopoietic cell line panel.

FIG. 5 shows survival of animals after dosing of peptide 1 in AML xenograft model.

FIG. 6 shows the dose-dependant platelet response of peptide 1 in 4-week monkey GLP toxicity study.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Definitions

As used herein, the term “macrocycle” refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” or “crosslinked polypeptide” refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker which forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule. Peptidomimetic macrocycle include embodiments where the macrocycle-forming linker connects the α carbon of the first amino acid residue (or analog) to the α carbon of the second amino acid residue (or analog). The peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog residues in addition to any which form the macrocycle. A “corresponding uncrosslinked polypeptide” when referred to in the context of a peptidomimetic macrocycle is understood to relate to a polypeptide of the same length as the macrocycle and comprising the equivalent natural amino acids of the wild-type sequence corresponding to the macrocycle.

As used herein, the term “laboratory TLS” refers to a 25% increase in the levels of serum uric acid, potassium, or phosphorus or a 25% decrease in calcium levels.

As used herein, the term “helical stability” refers to the maintenance of a helical structure by a peptidomimetic macrocycle as measured by circular dichroism or NMR. For example, in some embodiments, a peptidomimetic macrocycle exhibits at least a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as determined by circular dichroism compared to a corresponding uncrosslinked macrocycle.

The term “amino acid” refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. The term amino acid, as used herein, includes, without limitation, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

The term “α-amino acid” refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the α-carbon.

The term “β-amino acid” refers to a molecule containing both an amino group and a carboxyl group in a P configuration.

The term “naturally occurring amino acid” refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

The following table shows a summary of the properties of natural amino acids:

Side-chain Hydro- 3 -Letter 1-Letter Side-chain charge pathy Amino Acid Code Code Polarity (pH 7.4) Index Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive −4.5 Asparagine Asn N polar neutral −3.5 Aspartic acid Asp D polar negative −3.5 Cysteine Cys C polar neutral 2.5 Glutamic acid Glu E polar negative −3.5 Glutamine Gln Q polar neutral −3.5 Glycine Gly G nonpolar neutral −0.4 Histidine His H polar positive(10%) −3.2 neutral(90%) Isoleucine Ile I nonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys K polar positive −3.9 Methionine Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral −1.6 Serine Ser S polar neutral −0.8 Threonine Thr T polar neutral −0.7 Tryptophan Trp W nonpolar neutral −0.9 Tyrosine Tyr Y polar neutral −1.3 Valine Val V nonpolar neutral 4.2

“Hydrophobic amino acids” include small hydrophobic amino acids and large hydrophobic amino acids. “Small hydrophobic amino acid” are glycine, alanine, proline, and analogs thereof. “Large hydrophobic amino acids” are valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. “Polar amino acids” are serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. “Charged amino acids” are lysine, arginine, histidine, aspartate, glutamate, and analogs thereof.

The term “amino acid analog” refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, without limitation, β-amino acids and amino acids where the amino or carboxy group is substituted by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).

The term “non-natural amino acid” refers to an amino acid which is not one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. Non-natural amino acids or amino acid analogs include, without limitation, structures according to the following:

Amino acid analogs include β-amino acid analogs. Examples of β-amino acid analogs include, but are not limited to, the following: cyclic β-amino acid analogs; β-alanine; (R)-β-phenylalanine; (R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl)-butyric acid; (R)-3-amino-4-(2-furyl)-butyric acid; (R)-3-amino-4-(2-methylphenyl)-butyric acid; (R)-3-amino-4-(2-naphthyl)-butyric acid; (R)-3-amino-4-(2-thienyl)-butyric acid; (R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(3,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(3,4-difluorophenyl)butyric acid; (R)-3-amino-4-(3-benzothienyl)-butyric acid; (R)-3-amino-4-(3-chlorophenyl)-butyric acid; (R)-3-amino-4-(3-cyanophenyl)-butyric acid; (R)-3-amino-4-(3-fluorophenyl)-butyric acid; (R)-3-amino-4-(3-methylphenyl)-butyric acid; (R)-3-amino-4-(3-pyridyl)-butyric acid; (R)-3-amino-4-(3-thienyl)-butyric acid; (R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(4-bromophenyl)-butyric acid; (R)-3-amino-4-(4-chlorophenyl)-butyric acid; (R)-3-amino-4-(4-cyanophenyl)-butyric acid; (R)-3-amino-4-(4-fluorophenyl)-butyric acid; (R)-3-amino-4-(4-iodophenyl)-butyric acid; (R)-3-amino-4-(4-methylphenyl)-butyric acid; (R)-3-amino-4-(4-nitrophenyl)-butyric acid; (R)-3-amino-4-(4-pyridyl)-butyric acid; (R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-pentafluoro-phenylbutyric acid; (R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexynoic acid; (R)-3-amino-5-phenylpentanoic acid; (R)-3-amino-6-phenyl-5-hexenoic acid; (S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (S)-3-amino-4-(1-naphthyl)-butyric acid; (S)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (S)-3-amino-4-(2-chlorophenyl)-butyric acid; (S)-3-amino-4-(2-cyanophenyl)-butyric acid; (S)-3-amino-4-(2-fluorophenyl)-butyric acid; (S)-3-amino-4-(2-furyl)-butyric acid; (S)-3-amino-4-(2-methylphenyl)-butyric acid; (S)-3-amino-4-(2-naphthyl)-butyric acid; (S)-3-amino-4-(2-thienyl)-butyric acid; (S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(3,4-dichlorophenyl)butyric acid; (S)-3-amino-4-(3,4-difluorophenyl)butyric acid; (S)-3-amino-4-(3-benzothienyl)-butyric acid; (S)-3-amino-4-(3-chlorophenyl)-butyric acid; (S)-3-amino-4-(3-cyanophenyl)-butyric acid; (S)-3-amino-4-(3-fluorophenyl)-butyric acid; (S)-3-amino-4-(3-methylphenyl)-butyric acid; (S)-3-amino-4-(3-pyridyl)-butyric acid; (S)-3-amino-4-(3-thienyl)-butyric acid; (S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(4-bromophenyl)-butyric acid; (S)-3-amino-4-(4-chlorophenyl)-butyric acid; (S)-3-amino-4-(4-cyanophenyl)-butyric acid; (S)-3-amino-4-(4-fluorophenyl)-butyric acid; (S)-3-amino-4-(4-iodophenyl)-butyric acid; (S)-3-amino-4-(4-methylphenyl)-butyric acid; (S)-3-amino-4-(4-nitrophenyl)-butyric acid; (S)-3-amino-4-(4-pyridyl)-butyric acid; (S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-pentafluoro-phenylbutyric acid; (S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexynoic acid; (S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid; 1,2,5,6-tetrahydropyridine-4-carboxylic acid; 3-amino-3-(2-chlorophenyl)-propionic acid; 3-amino-3-(2-thienyl)-propionic acid; 3-amino-3-(3-bromophenyl)-propionic acid; 3-amino-3-(4-chlorophenyl)-propionic acid; 3-amino-3-(4-methoxyphenyl)-propionic acid; 3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid; D-β-phenylalanine; β-leucine; L-β-homoalanine; L-β-homoaspartic acid γ-benzyl ester; L-β-homoglutamic acid δ-benzyl ester; L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionine; L-β-homophenylalanine; L-β-homoproline; L-β-homotryptophan; L-β-homovaline; L-Nω-benzyloxycarbonyl-3-homolysine; Nω-L-β-homoarginine; O-benzyl-L-β-homohydroxyproline; O-benzyl-L-β-homoserine; O-benzyl-L-β-homothreonine; O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoasparagine; (R)-3-phenylalanine; L-β-homoaspartic acid γ-t-butyl ester; L-β-homoglutamic acid δ-t-butyl ester; L-Nω-3-homolysine; Nδ-trityl-L-β-homoglutamine; Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine; O-t-butyl-L-β-homohydroxy-proline; O-t-butyl-L-β-homoserine; O-t-butyl-L-β-homothreonine; O-t-butyl-L-β-homotyrosine; 2-aminocyclopentane carboxylic acid; and 2-aminocyclohexane carboxylic acid.

Amino acid analogs include analogs of alanine, valine, glycine or leucine. Examples of amino acid analogs of alanine, valine, glycine, and leucine include, but are not limited to, the following: α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid; α-methyl-leucine; β-(1-naphthyl)-D-alanine; β-(1-naphthyl)-L-alanine; β-(2-naphthyl)-D-alanine; β-(2-naphthyl)-L-alanine; β-(2-pyridyl)-D-alanine; β-(2-pyridyl)-L-alanine; β-(2-thienyl)-D-alanine; β-(2-thienyl)-L-alanine; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; β-(3-pyridyl)-D-alanine; β-(3-pyridyl)-L-alanine; β-(4-pyridyl)-D-alanine; β-(4-pyridyl)-L-alanine; β-chloro-L-alanine; β-cyano-L-alanin; β-cyclohexyl-D-alanine; β-cyclohexyl-L-alanine; β-cyclopenten-1-yl-alanine; β-cyclopentyl-alanine; β-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt; β-t-butyl-D-alanine; β-t-butyl-L-alanine; γ-aminobutyric acid; L-α,β-diaminopropionic acid; 2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine; 2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine; 3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine; 4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine; 4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic acid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt; cyclopentyl-Gly-OH.dicyclohexylammonium salt; D-α,β-diaminopropionic acid; D-α-aminobutyric acid; D-α-t-butylglycine; D-(2-thienyl)glycine; D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine; D-allylglycine.dicyclohexylammonium salt; D-cyclohexylglycine; D-norvaline; D-phenylglycine; β-aminobutyric acid; 3-aminoisobutyric acid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine; (2-methylphenyl)glycine; (2-thiazoyl)glycine; (2-thienyl)glycine; 2-amino-3-(dimethylamino)-propionic acid; L-α,β-diaminopropionic acid; L-α-aminobutyric acid; L-α-t-butylglycine; L-(3-thienyl)glycine; L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic acid dicyclohexyl-ammonium salt; L-2-indanylglycine; L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine; L-phenylglycine; L-propargylglycine; L-norvaline; N-α-aminomethyl-L-alanine; D-α,γ-diaminobutyric acid; L-α,γ-diaminobutyric acid; 0-cyclopropyl-L-alanine; (N-3-(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,β-diaminopropionic acid; (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid; (N-β-allyloxycarbonyl)-L-α,β-diaminopropionic acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyric acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid; (N-γ-allyloxycarbonyl)-L-α,γ-diaminobutyric acid; D-α,γ-diaminobutyric acid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; and N-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.

Amino acid analogs include analogs of arginine or lysine. Examples of amino acid analogs of arginine and lysine include, but are not limited to, the following: citrulline; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me)₂-OH; Lys(N₃)—OH; Nδ-benzyloxycarbonyl-L-ornithine; Nco-nitro-D-arginine; Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminoheptanedioic acid; L-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-omithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-omithine; (Nδ-4-methyltrityl)-D-ornithine; (Nδ-4-methyltrityl)-L-omithine; D-omithine; L-ornithine; Arg(Me)(Pbf)-OH; Arg(Me)₂-OH (asymmetrical); Arg(Me)₂-OH (symmetrical); Lys(ivDde)-OH; Lys(Me)₂-OH.HCl; Lys(Me3)-OH chloride; Nco-nitro-D-arginine; and Nω-nitro-L-arginine.

Amino acid analogs include analogs of aspartic or glutamic acids. Examples of amino acid analogs of aspartic and glutamic acids include, but are not limited to, the following: α-methyl-D-aspartic acid; α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamic acid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelic acid; L-α-aminosuberic acid; D-2-aminoadipic acid; D-α-aminosuberic acid; α-aminopimelic acid; iminodiacetic acid; L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamic acid γ,γ-di-t-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-t-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and pyroglutamic acid.

Amino acid analogs include analogs of cysteine and methionine. Examples of amino acid analogs of cysteine and methionine include, but are not limited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, α-methyl-methionine, Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH, 2-amino-4-(ethylthio)butyric acid, buthionine, buthioninesulfoximine, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine, 4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine, carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine, t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine, Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.

Amino acid analogs include analogs of phenylalanine and tyrosine. Examples of amino acid analogs of phenylalanine and tyrosine include β-methyl-phenylalanine, β-hydroxyphenylalanine, α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine, α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine, 2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine, 2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine, 3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine, 3,5,3′-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine, 3-(trifluoromethyl)-D-phenylalanine, 3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine, 3-nitro-L-phenylalanine, 3-nitro-L-tyrosine, 4-(trifluoromethyl)-D-phenylalanine, 4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine, 4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine, 4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine, thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tyrosine, and methyl-tyrosine.

Amino acid analogs include analogs of proline. Examples of amino acid analogs of proline include, but are not limited to, 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.

Amino acid analogs include analogs of serine and threonine. Examples of amino acid analogs of serine and threonine include, but are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid, 2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid, and α-methylserine.

Amino acid analogs include analogs of tryptophan. Examples of amino acid analogs of tryptophan include, but are not limited to, the following: α-methyl-tryptophan; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan; 7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid; 7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

In some embodiments, amino acid analogs are racemic. In some embodiments, the D isomer of the amino acid analog is used. In some embodiments, the L isomer of the amino acid analog is used. In other embodiments, the amino acid analog comprises chiral centers that are in the R or S configuration. In still other embodiments, the amino group(s) of a β-amino acid analog is substituted with a protecting group, e.g., tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like. In yet other embodiments, the carboxylic acid functional group of a β-amino acid analog is protected, e.g., as its ester derivative. In some embodiments the salt of the amino acid analog is used.

A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation). An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted nonessential amino acid residue in a polypeptide, for example, is replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine, or 6-Cl-tryptophan for tryptophan).

The term “capping group” refers to the chemical moiety occurring at either the carboxy or amino terminus of the polypeptide chain of the subject peptidomimetic macrocycle. The capping group of a carboxy terminus includes an unmodified carboxylic acid (i.e. —COOH) or a carboxylic acid with a substituent. For example, the carboxy terminus can be substituted with an amino group to yield a carboxamide at the C-terminus. Various substituents include but are not limited to primary and secondary amines, including pegylated secondary amines. Representative secondary amine capping groups for the C-terminus include:

The capping group of an amino terminus includes an unmodified amine (ie-NH₂) or an amine with a substituent. For example, the amino terminus can be substituted with an acyl group to yield a carboxamide at the N-terminus. Various substituents include but are not limited to substituted acyl groups, including C₁-C₆ carbonyls, C₇-C₃₀ carbonyls, and pegylated carbamates. Representative capping groups for the N-terminus include, but are not limited to, 4-FBzl (4-fluoro-benzyl) and the following:

The term “member” as used herein in conjunction with macrocycles or macrocycle-forming linkers refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.

The symbol “

” when used as part of a molecular structure refers to a single bond or a trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to the α-carbon (or another backbone atom) in an amino acid. For example, the amino acid side chain for alanine is methyl, the amino acid side chain for phenylalanine is phenylmethyl, the amino acid side chain for cysteine is thiomethyl, the amino acid side chain for aspartate is carboxymethyl, the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature (e.g., an amino acid metabolite) or those that are made synthetically (e.g., an α,αdi-substituted amino acid).

The term “α,αdi-substituted amino” acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to a carbon (the α-carbon) that is attached to two natural or non-natural amino acid side chains.

The term “polypeptide” encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).

The term “first C-terminal amino acid” refers to the amino acid which is closest to the C-terminus. The term “second C-terminal amino acid” refers to the amino acid attached at the N-terminus of the first C-terminal amino acid.

The term “macrocyclization reagent” or “macrocycle-forming reagent” as used herein refers to any reagent which can be used to prepare a peptidomimetic macrocycle by mediating the reaction between two reactive groups. Reactive groups can be, for example, an azide and alkyne, in which case macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II) salts such as Cu(CO₂CH₃)₂, CuSO₄, and CuCl₂ that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate. Macrocyclization reagents can additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh₃)₂, [Cp*RuCl]₄ or other Ru reagents which can provide a reactive Ru(II) species. In other cases, the reactive groups are terminal olefins. In such embodiments, the macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts. For example, such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and pentacoordinated. In other examples, catalysts have W or Mo centers. Various catalysts are disclosed in Grubbs et al., “Ring Closing Metathesis and Related Processes in Organic Synthesis” Acc. Chem. Res. 1995, 28, 446-452, U.S. Pat. Nos. 5,811,515; 7,932,397; U.S. Application No. 2011/0065915; U.S. Application No. 2011/0245477; Yu et al., “Synthesis of Macrocyclic Natural Products by Catalyst-Controlled Stereoselective Ring-Closing Metathesis,” Nature 2011, 479, 88; and Peryshkov et al., “Z-Selective Olefin Metathesis Reactions Promoted by Tungsten Oxo Alkylidene Complexes,” J. Am. Chem. Soc. 2011, 133, 20754. In yet other cases, the reactive groups are thiol groups. In such embodiments, the macrocyclization reagent is, for example, a linker functionalized with two thiol-reactive groups such as halogen groups. In some examples, the macrocyclization reagent include palladium reagents, for example Pd(PPh₃)₄, Pd(PPh₃)₂Cl₂, Pd(dppe)Cl, Pd(dppp)Cl₂, and Pd(dppf)Cl₂.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine or iodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive) carbon atoms in it. In the absence of any numerical designation, “alkyl” is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon double bonds. The alkenyl moiety contains the indicated number of carbon atoms. For example, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenyl chain. In the absence of any numerical designation, “alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon triple bonds. The alkynyl moiety contains the indicated number of carbon atoms. For example, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynyl chain. In the absence of any numerical designation, “alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term “arylalkoxy” refers to an alkoxy substituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkyl group, as defined above. Representative examples of an arylalkyl group include, but are not limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with one or more —C(O)NH₂ groups. Representative examples of an arylamido group include 2-C(O)NH₂-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl, 3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl.

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above, wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a heterocycle. Representative examples of an alkylheterocycle group include, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine, —CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a —C(O)NH₂ group. Representative examples of an alkylamido group include, but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂, —CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃, —C(CH₃)₂CH₂C(O)NH₂, —CH₂—CH₂—NH—C(O)—CH₃, —CH₂—CH₂—NH—C(O)—CH₃—CH₃, and —CH₂—CH₂—NH—C(O)—CH═CH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a hydroxyl group. Representative examples of an alkanol group include, but are not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and —C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a —COOH group. Representative examples of an alkylcarboxy group include, but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH, —CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted. Some cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring are substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom or group such as a hydrogen atom on any molecule, compound or moiety. Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds disclosed herein contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are included unless expressly provided otherwise. In some embodiments, the compounds disclosed herein are also represented in multiple tautomeric forms, in such instances, the compounds include all tautomeric forms of the compounds described herein (e.g., if alkylation of a ring system results in alkylation at multiple sites, the disclosure includes all such reaction products). All such isomeric forms of such compounds are included unless expressly provided otherwise. All crystal forms of the compounds described herein are included unless expressly provided otherwise.

As used herein, the terms “increase” and “decrease” mean, respectively, to cause a statistically significantly (i.e., p<0.1) increase or decrease of at least 5%.

As used herein, the recitation of a numerical range for a variable is intended to convey that the variable is equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable is equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable is equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 takes the values 0, 1 or 2 if the variable is inherently discrete, and takes the values 0.0, 0.1, 0.01, 0.001, or any other real values ≥0 and ≤2 if the variable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or.”

The term “on average” represents the mean value derived from performing at least three independent replicates for each data point.

The term “biological activity” encompasses structural and functional properties of a macrocycle. Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell penetrability, intracellular stability, in vivo stability, or any combination thereof.

The term “binding affinity” refers to the strength of a binding interaction, for example between a peptidomimetic macrocycle and a target. Binding affinity can be expressed, for example, as an equilibrium dissociation constant (“K_(D)”), which is expressed in units which are a measure of concentration (e.g. M, mM, μM, nM etc). Numerically, binding affinity and K_(D) values vary inversely, such that a lower binding affinity corresponds to a higher K_(D) value, while a higher binding affinity corresponds to a lower K_(D) value. Where high binding affinity is desirable, “improved” binding affinity refers to higher binding affinity and therefore lower K_(D) values.

The term “in vitro efficacy” refers to the extent to which a test compound, such as a peptidomimetic macrocycle, produces a beneficial result in an in vitro test system or assay. In vitro efficacy can be measured, for example, as an “IC₅₀” or “EC₅₀” value, which represents the concentration of the test compound which produces 50% of the maximal effect in the test system.

The term “ratio of in vitro efficacies” or “in vitro efficacy ratio” refers to the ratio of IC₅₀ or EC₅₀ values from a first assay (the numerator) versus a second assay (the denominator). Consequently, an improved in vitro efficacy ratio for Assay 1 versus Assay 2 refers to a lower value for the ratio expressed as IC₅₀(Assay 1)/IC₅₀(Assay 2) or alternatively as EC₅₀(Assay 1)/EC₅₀(Assay 2). This concept can also be characterized as “improved selectivity” in Assay 1 versus Assay 2, which can be due either to a decrease in the IC₅₀ or EC₅₀ value for Target 1 or an increase in the value for the IC₅₀ or EC₅₀ value for Target 2.

The term “liquid cancer” as used herein refers to cancer cells that are present in body fluids, such as blood, lymph and bone marrow. Liquid cancers include leukemia, myeloma, myelodysplastic syndrome (MDS), and liquid lymphomas. For example, liquid cancer can be acute myeloid leukemia (AML). Liquid lymphomas include lymphomas that contain cysts or liquid areas. Liquid cancers as used herein do not include solid tumors, such as sarcomas and carcinomas or solid lymphomas that do not contain cysts or liquid areas.

The term “adverse event” (AE) as used herein includes any noxious, pathological, or unintended change in anatomical, physiological, or metabolic functions as indicated by physical signs, symptoms, and/or laboratory changes occurring in any phase of the clinical study whether or not temporally associated with the administration of study medication and whether or not considered related to the study medication. This definition includes an exacerbation of pre-existing medical conditions or events, intercurrent illnesses, hypersensitivity reactions, drug interactions, or clinically significant laboratory findings. An AE does not include the following: (i) medical or surgical procedures, e.g., tooth extraction, transfusion, surgery (The medical condition that leads to the procedure is to be recorded as an AE); (ii) pre-existing conditions or procedures present or detected at the start of the study that do not worsen; (iii) hospitalization for elective surgeries or for other situations in which an untoward medical event has not occurred; (iv) abnormal laboratory value, unless it is clinically significant according to the Investigator, requires intervention, or results in a delay, discontinuation or change in the dose of study drug; (v) overdose of study drug or concomitant medication unaccompanied by signs/symptoms; if sign/symptoms occur, the final diagnosis should be recorded as an AE; (vi) pregnancy by itself, unless a complication occurs during pregnancy leading to hospitalization; in this case, the medical condition that leads to the hospitalization is to be recorded as the AE; and (vii) significant worsening of the disease under investigation which is captured as an efficacy parameter in this study and, thus, is not recorded as an AE.

The term serious adverse event (SAE) as used herein refers to an adverse event that results in any of the following outcomes: (i) death; (ii) life-threatening adverse experience (i.e., immediate risk of death from the event as it occurred; this does not include an adverse event that, had it occurred in a more serious form, might have caused death); (iii) persistent or significant disability/incapacitation; (iv) hospitalization or prolongation of existing hospitalization; and (v) congenital anomaly/birth defect.Important medical events that can not result in death, be life-threatening, or require hospitalization can be considered serious when, based on medical judgment, they can jeopardize the patient or can require medical or surgical intervention to prevent one of the outcomes listed in this definition. Hospitalizations due to the underlying disease will not be reported as an SAE unless there is reason to suspect a causal relationship with the study drug.

An AE or suspected adverse reaction is considered “unexpected” (referred to as Unexpected Adverse Event (UAE) if it is not listed in the peptidomimetic macrocycle Investigator's Brochure or is not listed at the specificity or severity that has been observed; or, is not consistent with the risk information described in the protocol or elsewhere. For example, under this definition, hepatic necrosis would be unexpected (by virtue of greater severity) if the Investigator's Brochure referred only to elevated hepatic enzymes or hepatitis. Similarly, cerebral thromboembolism and cerebral vasculitis would be unexpected (by virtue of greater specificity) if the Investigator's Brochure listed only cerebral vascular accidents. “Unexpected,” as used in this definition, also refers to AEs or suspected adverse reactions that are mentioned in the Investigator's Brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the peptidomimetic macrocycle but are not specifically mentioned as occurring with the peptidomimetic macrocycle.

A “Dose-Limiting Toxicity” (DLT) as used herein is defined as any non hematologic Grade ≥3 AE that is considered to be possibly, probably, or definitely related to the study drug, with the following exceptions: (1) for fatigue, nausea, emesis, diarrhea or mucositis, all Grade 4 and any Grade 3 AE requiring total parenteral nutrition (TPN) or hospitalization will be considered DLT; (2) for electrolyte imbalances, only Grade ≥3 AE that do not respond to correction within 24 hours will be considered DLT; (3) for infusion reactions, only a Grade 3 reaction which caused hospitalization or Grade 4 will be considered DLT; (4) any grade alopecia; (5) any event that can clearly be determined to be unrelated to the study drug (e.g., solely related to disease progression). DLT also includes: i) ANC fails to recover to >0.5 Gi/L within 42 days from the start of therapy in the absence of active leukemia or myelodysplasia; and ii) Platelet count fails to recover to >20,000 or associated with clinically significant bleeding that requires transfusion of red cells or platelets within 42 days from the start of therapy in the absence of active leukemia or myelodysplasia. In addition, specific hematologic DLTs are defined as:

-   -   (i) Thrombocytopenia-Grade 4 of any duration, Grade 3 for ≥7         days, or Grade 3 associated with clinically significant         bleeding;     -   (ii) Neutropenia-Grade 4 for ≥3 days, or any Grade ≥3 febrile         neutropenia.

The above criteria can be used to make individual patient determinations regarding dose reductions, interruptions or discontinuation throughout the course of the trial, but DLTs occurring during Cycle 1 will be used to inform safety and tolerability assessments for dose escalation decisions. The DLT-evaluable population will include all patients in Phase 1 Dose Escalation who experience a DLT during the first cycle of treatment.

The “Maximum Tolerated Dose” (MTD) as used herein is defined as the dose at which ≤1 of 6 patients experiences a treatment-related toxicity that qualifies as a DLT, with the next higher dose having ≥2 of up to 6 patients experiencing a DLT. The MTD can not be established until all patients enrolled in the cohort have completed Cycle 1, discontinued treatment or had a dose reduction. Previously established tolerability of a dose level will be reevaluated if DLTs are observed in later cycles.

The term “subject” or “patient” encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, humans; non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

The details of one or more particular embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Overview

In one aspect, the disclosure provides a method of treating liquid cancer, determined to lack a p53 deactivating mutation, in a subject. The method comprises administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In some embodiments, the peptidomimetic macrocycle disrupts the interaction between p53 and MDM2 and MDMX.

In another aspect, the disclosure provides a method of treating liquid cancer in a subject expressing wild type p53. The method comprises administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In some embodiments, the peptidomimetic macrocycle disrupts the interaction between p53 and MDM2 and MDMX.

In some embodiments, a subject treated in accordance with the methods provided herein is a human who has or is diagnosed with liquid cancer lacking p53 deactivating mutation and/or expressing wild type p53. In some embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human predisposed or susceptible to liquid cancer lacking p53 deactivating mutation and/or expressing wild type p53. In some embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human at risk of developing liquid cancer lacking p53 deactivating mutation and/or expressing wild type p53. A p53 deactivating mutation in some example can be a mutation in DNA-binding domain of the p53 protein. In some examples the p53 deactivating mutation can be a missense mutation. In various examples, the liquid cancer can be determined to lack one or more p53 deactivating mutations selected from mutations at one or more of residues R175, G245, R248, R249, R273, and R282. The lack of p53 deactivating mutation and/or the presence of wild type p53 in the liquid cancer can be determined by any suitable method known in art, for example by sequencing, array based testing, RNA analysis and amplifications methods like PCR.

In certain embodiments, the human subject is refractory and/or intolerant to one or more other standard treatment of the liquid cancer known in art. In some embodiments, the human subject has had at least one unsuccessful prior treatment and/or therapy of the liquid cancer.

In some embodiments, the methods for treating liquid cancer provided herein inhibit, reduce, diminish, arrest, or stabilize a liquid cancer cell associated with the liquid cancer. In some embodiments, the methods for treating liquid cancer provided herein inhibit, reduce, diminish, arrest, or stabilize the blood flow, metabolism, or edema in a liquid cancer cell associated with the liquid cancer or one or more symptoms thereof. In some embodiments, the methods for treating liquid cancer provided herein cause the regression of the number od liquid cancer cells, or liquid cancer cell metabolism, and/or one or more symptoms associated with the liquid cancer. In some embodiments, the methods for treating liquid cancer provided herein maintain the number of CTCs and/or MNBCs so that the number of CTCs and/orMNBCs does not increase, or increases by less than the increase of the number of CTCs and/or MNBCs after administration of a standard therapy as measured by conventional methods available to one of skill in the art, such as ultrasound, CT Scan, MRI, dynamic contrast-enhanced MRI, or PET Scan. In specific embodiments, the methods for treating liquid cancer provided herein decrease liquid cancer cell number. In some embodiments, the methods for treating liquid cancer provided herein reduce the formation of CTCs and/or MNBCs. In certain embodiments, the methods for treating liquid cancer provided herein eradicate, remove, or control primary, regional and/or metastatic liquid cancer cells associated with the liquid cancer. In some embodiments, the methods for treating liquid cancer provided herein decrease the number or size of metastases associated with the liquid cancer. In some embodiments, the methods for treating liquid cancer provided herein result in complete response to the treatment. In some embodiments, the methods for treating liquid cancer provided herein result in partial response to the treatment. In some embodiments, the liquid cancer treated by the methods disclosed herein is a stable disease. In some embodiments, the liquid cancer treated by the methods disclosed herein is a progressive disease.

Liquid cancer cancers that can be treated by the methods provided herein include, but are not limited to, leukemias, myelomas, and liquid lymphomas. In specific embodiments, liquid cancers that can be treated in accordance with the methods described include, but are not limited to, liquid lymphomas, lekemias, and myelomas. Exemplary liquid lymphomas and leukemias that can be treated in accordance with the methods described include, but are not limited to, acute myelogenous leukemia (AML), myelodysplastic syndromes (MDS), chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma, classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte depleted or not depleted), and nodular lymphocyte-predominant Hodgkin lymphoma.

In one aspect, myelodysplastic syndromes (MDS) is a heterogenous group of clonal, hematopoietic stem cell disorders characterized by distinct morphological bone marrow changes, abnormal blood counts, common cytogenetic abnormalities, and recurrent mutations. MDS can predominantly occur in the elderly. Treatment of MDS can be based on risk stratification, with the International Prognostic Scoring System (IPSS) or revised IPSS (IPSS-R) being the most common classification systems. Low-risk MDS patients can receive supportive care or hematopoietic growth factors. A subset of patients with 5q deletions can be treated with lenalidomide. High-risk patients can be treated with hypomethylating agents (e.g., azacitidine, decitabine), intensive chemotherapy, and/or allogeneic stem cell transplantation. In some cases, MDS patients can be transformed to AML. Some MDS patients can develop progressive bone marrow failure and/or die of complications related to neutropenia (e.g., infection) or thrombocytopenia (e.g., bleeding). Initial management of MDS can be based on risk stratification. The newer IPSS-R can place patients into 5 categories: very good, good, intermediate, high, and very-high risk groups. Patients in the very good, good, and select intermediate-risk patients can be categorized as “low-risk,” whereas high, very high, and certain intermediate-risk patients can be categorized as the “high-risk” group. Azacitidine (5′-azacytidine) and decitabine (5′-aza-2′-deoxycytidine), which both are cytosine analogues, can lead to inhibition of DNA-methyltransferases (DNMTs) and can act as hypomethylating agents.

In another aspect, acute myeloid leukemia (AML) is characterized by the proliferation and accumulation of myeloid cells with accompanying hematopoietic failure. AML can be caused by chemical exposure, prior chemotherapy and radiation, or other environmental toxins.

Examples of liquid cancers includes cancers involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Exemplary disorders include: acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), multiple mylenoma, hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant liquid lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease. For example, liquid cancers include, but are not limited to, acute lymphocytic leukemia (ALL); T-cell acute lymphocytic leukemia (T-ALL); anaplastic large cell lymphoma (ALCL); chronic myelogenous leukemia (CML); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); B-cell chronic lymphocytic leukemia (B-CLL); diffuse large B-cell lymphomas (DLBCL); hyper eosinophilia/chronic eosinophilia; and Burkitt's lymphoma.

In embodiments, the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; AIDS-related cancers; AIDS-related lymphoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; cutaneous T-cell lymphoma; Hodgkin lymphoma; multiple myeloma; multiple myeloma/plasma cell neoplasm; Non-Hodgkin lymphoma; primary central nervous system (CNS) lymphoma; or T-cell lymphoma; In various embodiments, the liquid cancer can be B-Cell Chronic Lymphocytic Leukemia, B-Cell Lymphoma-DLBCL, B-Cell Lymphoma-DLBCL-germinal center-like, B-Cell Lymphoma-DLBCL-activated B-cell-like, or Burkitt's lymphoma.

The peptidomimetic macrocycle can be any cross-linked peptide, i.e. any peptide that comprises at least one macrocycle-forming linker which forms a macrocycle between a first amino acid residue (or analog) and a second amino acid residue. For example, the peptidomimetic macrocycle can be a peptidomimetic macrocycle capable of binding to the MDM2 and/or MDMX proteins. In some embodiments, the peptidomimetic macrocycles can be a peptidomimetic macrocycle of Formula I:

wherein: each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₅, Xaa₉, and Xaa₁₀ is individually an amino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₅, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Alas-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8) or Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9), where each X is an amino acid; each D and E is independently an amino acid; R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids; each L or L′ is independently a macrocycle-forming linker; each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue; R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue; v is an integer from 1-1000, for example 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, or 1-10; and w is an integer from 0-1000.

Administration of the peptidomimetic macrocycle can be achieved by any suitable means. For example the peptidomimetic macrocycle can be administered parenterally. For example, administration can be intravenous, intra-arterial, intraosseous infusion, intra-muscular, intracerebral, intracerebroventricular, intrathecal, or subcutaneous. In some embodiments administration is performed intravenously.

In some embodiments, the methods disclosed herein additionally or optionally comprise evaluating the safety and/or tolerability of the peptidomimetic macrocycles of the disclosure in subjects with liquid cancers determined to lack a p53 deactivating mutation or with liquid cancers expressing wild-type (WT) p53 protein.

Also provided here in are methods to determine the dose limiting toxicities (DLT) and the maximum tolerated dose (MTD or OBD) or the optimal biological dose (OBD) of the peptidomimetic macrocycles disclosed herein in subjects with liquid cancers determined to lack a p53 deactivating mutation or with liquid cancers expressing wild-type (WT) p53 protein.

In some embodiments, the methods disclosed herein additionally or optionally comprise the pharmacokinetic (PK) analysis of the peptidomimetic macrocycles and/or its metabolites in blood following single and/or multiple administration of the peptidomimetic macrocycles to the subject.

In some embodiments, the methods disclosed herein additionally or optionally comprise studying the effect of the peptidomimetic macrocycles on pharmacodynamic (PD) biomarkers in liquid cancer samples (including bone marrow aspirates), (e.g., p21, caspase, MDM2) and blood samples (e.g., macrophage inhibitory cytokine-1 [MIC-1]), and assessing possible correlation between these biomarkers and clinical response.

In some embodiments, the methods disclosed herein additionally or optionally include steps to assess potential patient biomarkers (e.g., p53 status, MDM2 and MDMX expression levels), the effect of the peptidomimetic macrocycles treatment on these biomarkers, and possible correlation between these biomarkers and clinical response of the peptidomimetic macrocycles.

Also provided herein are methods to evaluate clinical activity of the peptidomimetic macrocycles in subjects with specific liquid cancer types lacking a p53 deactivating mutation and/or expressing WT p53 in the dose expansion phase.

Compound and Compositions Peptidomimetic Macrocycles

In some embodiments, a peptidomimetic macrocycle has the Formula (I):

wherein: each A, C, and D is independently an amino acid; each B is independently an amino acid,

[—N-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-]; each E is independently an amino acid selected from the group consisting of Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine); each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅; each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids; each L and L′ is independently a macrocycle-forming linker; each L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionally substituted with R₅; each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃; each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue; each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue; each v is independently an integer from 0-1000, for example, 0-500, 0-200, 0-100, 0-50, 0-30, 0-20, 0-10, 0-5, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, 1-10, 1-5, 3-1000, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each w is independently an integer from 0-1000, for example, 0-500, 0-200, 0-100, 0-50, 0-30, 0-20, 0-10, 0-5, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, 1-10, 1-5, 3-1000, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; u is an integer from 1-10; each x, y and z is independently an integer from 0-10; and each n is independently an integer from 1-5.

In some embodiments, each v and w is independently integers between 1-30. In some embodiments, w or v is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6.

In some embodiments, peptidomimetic macrocycles are also provided of the formula:

wherein: each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually an amino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8), where each X is an amino acid; each D and E is independently an amino acid; each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids or Xaa₃; each L or L′ is independently a macrocycle-forming linker; each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue; each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue; v is an integer from 0-1000, for example, 0-500, 0-200, 0-100, 0-50, 0-30, 0-20, 0-10, 0-5, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, 1-10, 1-5, 3-1000, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and w is an integer from 0-1000, for example, 0-500, 0-200, 0-100, 0-50, 0-30, 0-20, 0-10, 0-5, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, 1-10, 1-5, 3-1000, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, each v and w is independently an integer between 1-30. In some embodiments, w or v is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6.

In some embodiments of any of the Formulas described herein, at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8). In other embodiments, at least four of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8). In other embodiments, at least five of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8). In other embodiments, at least six of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8). In other embodiments, at least seven of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8).

In some embodiments, a peptidomimetic macrocycle has the Formula:

wherein: each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually an amino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9), wherein each X is an amino acid; each D is independently an amino acid; each E is independently an amino acid, for example an amino acid selected from Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine); each R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids; each L or L′ is independently a macrocycle-forming linker; each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue; each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue; v is an integer from 0-1000, for example, 0-500, 0-200, 0-100, 0-50, 0-30, 0-20, 0-10, 0-5, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, 1-10, 1-5, 3-1000, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and w is an integer from 0-1000, for example, 0-500, 0-200, 0-100, 0-50, 0-30, 0-20, 0-10, 0-5, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, 1-10, 1-5, 3-1000, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments of the above Formula, at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Alas-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9). In other embodiments of the above Formula, at least four of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9). In other embodiments of the above Formula, at least five of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Alas-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9). In other embodiments of the above Formula, at least six of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9). In other embodiments of the above Formula, at least seven of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9).

In some embodiments, w is an integer from 3-10, for example 3-6, 3-8, 6-8, or 6-10. In some embodiments, w is 3. In other embodiments, w is 6. In some embodiments, v is an integer from 1-10, for example 2-5. In some embodiments, v is 2.

In an embodiment of any of the Formulas described herein, of the macrocycle-forming linker (L) has a formula-L₁-L₂-, wherein L₁ and L₂ are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionally substituted with R₅;

each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃; each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅; and n is an integer from 1-5.

In some embodiments in the Formulas described herein, L (or L′) is a macrocycle-forming linker of the formula

Exemplary embodiments of such macrocycle-forming linkers L are shown below.

In an embodiment of any of the Formulas described herein, L₁ and L₂, either alone or in combination, form a triazole or a thioether.

In an embodiment of any of the Formulas described herein, L₁ and L₂, either alone or in combination, do not form a triazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted or substituted with halo-. In another example, both R₁ and R₂ are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R₁ and R₂ is methyl. In other embodiments, R₁ and R₂ are methyl.

In some embodiments, x+y+z is at least 3. In other embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor is independently selected. For example, a sequence represented by the formula [A]_(x), when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges. Similarly, when u is greater than 1, each compound can encompass peptidomimetic macrocycles which are the same or different. For example, a compound can comprise peptidomimetic macrocycles comprising different linker lengths or chemical compositions.

In some embodiments, the peptidomimetic macrocycle comprises a secondary structure which is an α-helix and R₈ is —H, allowing intrahelical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as an α-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.

In one embodiment, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁′ and R₂′ is independently an amino acid.

In other embodiments, the peptidomimetic macrocycle of Formula (I) is a compound of any of the formulas shown below:

wherein “AA” represents any natural or non-natural amino acid side chain and “

” is [D]_(v), [E]_(w) as defined above, and n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In other embodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shown below.

In other embodiments, D and/or E in the compound of Formula I are further modified in order to facilitate cellular uptake. In some embodiments, lipidating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.

In other embodiments, at least one of [D] and [E] in the compound of Formula I represents a moiety comprising an additional macrocycle-forming linker such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers. In a specific embodiment, a peptidomimetic macrocycle comprises two macrocycle-forming linkers. In an embodiment, u is 2.

In some embodiments, any of the macrocycle-forming linkers described herein can be used in any combination with any of the sequences shown in Table 3, Table 3a, Table 3b, or Table 3c and also with any of the R-substituents indicated herein.

In some embodiments, the peptidomimetic macrocycle comprises at least one α-helix motif. For example, A, B and/or C in the compound of Formula I include one or more α-helices. As a general matter, α-helices include between 3 and 4 amino acid residues per turn. In some embodiments, the α-helix of the peptidomimetic macrocycle includes 1 to 5 turns and, therefore, 3 to 20 amino acid residues. In specific embodiments, the α-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In some embodiments, the macrocycle-forming linker stabilizes an α-helix motif included within the peptidomimetic macrocycle. Thus, in some embodiments, the length of the macrocycle-forming linker L from a first Cα to a second Cα is selected to increase the stability of an α-helix. In some embodiments, the macrocycle-forming linker spans from 1 turn to 5 turns of the α-helix. In some embodiments, the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the α-helix. In some embodiments, the length of the macrocycle-forming linker is approximately 5 Å to 9 Å per turn of the α-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Where the macrocycle-forming linker spans approximately 1 turn of an α-helix, the length is equal to approximately 5 carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 2 turns of an α-helix, the length is equal to approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 3 turns of an α-helix, the length is equal to approximately 14 carbon-carbon bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 4 turns of an α-helix, the length is equal to approximately 20 carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 5 turns of an α-helix, the length is equal to approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 1 turn of an α-helix, the linkage contains approximately 4 atoms to 12 atoms, approximately 6 atoms to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming linker spans approximately 2 turns of the α-helix, the linkage contains approximately 7 atoms to 15 atoms, approximately 9 atoms to 13 atoms, or approximately 11 atoms. Where the macrocycle-forming linker spans approximately 3 turns of the α-helix, the linkage contains approximately 13 atoms to 21 atoms, approximately 15 atoms to 19 atoms, or approximately 17 atoms. Where the macrocycle-forming linker spans approximately 4 turns of the α-helix, the linkage contains approximately 19 atoms to 27 atoms, approximately 21 atoms to 25 atoms, or approximately 23 atoms. Where the macrocycle-forming linker spans approximately 5 turns of the α-helix, the linkage contains approximately 25 atoms to 33 atoms, approximately 27 atoms to 31 atoms, or approximately 29 atoms. Where the macrocycle-forming linker spans approximately 1 turn of the α-helix, the resulting macrocycle forms a ring containing approximately 17 members to 25 members, approximately 19 members to 23 members, or approximately 21 members. Where the macrocycle-forming linker spans approximately 2 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 29 members to 37 members, approximately 31 members to 35 members, or approximately 33 members. Where the macrocycle-forming linker spans approximately 3 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 44 members to 52 members, approximately 46 members to 50 members, or approximately 48 members. Where the macrocycle-forming linker spans approximately 4 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 59 members to 67 members, approximately 61 members to 65 members, or approximately 63 members. Where the macrocycle-forming linker spans approximately 5 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 74 members to 82 members, approximately 76 members to 80 members, or approximately 78 members.

In other embodiments, provided are peptidomimetic macrocycles of Formula (IV) or (IVa):

wherein: each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group; B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-]; R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids; R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅; L is a macrocycle-forming linker of the formula-L₁-L₂-; L₁, L₂ and L₃ are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionally substituted with R₅; each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is O, S, SO, SO₂, CO, CO₂, or CONR₃; each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅; v and w are independently integers from 1-1000; u is an integer from 1-10; x, y and z are independently integers from 0-10; and n is an integer from 1-5.

In one example, L₁ and L₂, either alone or in combination, do not form a triazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted or substituted with halo-. In another example, both R₁ and R₂ are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R₁ and R₂ is methyl. In other embodiments, R₁ and R₂ are methyl.

In some embodiments, x+y+z is at least 1. In other embodiments, x+y+z is at least 2. In other embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor is independently selected. For example, a sequence represented by the formula [A]_(x), when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.

In some embodiments, the peptidomimetic macrocycle comprises a secondary structure which is an α-helix and R₈ is —H, allowing intrahelical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as an α-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker -L₁-L₂- are shown below.

Unless otherwise stated, any compounds (including peptidomimetic macrocycles, macrocycle precursors, and other compositions) are also meant to encompass compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the described structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are within the scope of this disclosure.

In some embodiments, the compounds disclosed herein can contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds can be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). In other embodiments, one or more carbon atoms is replaced with a silicon atom. All isotopic variations of the compounds disclosed herein, whether radioactive or not, are contemplated herein.

The circulating half-life of the peptidomimetic macrocycles in human blood can be about 1-24 h. For example the circulating half-life of the peptidomimetic macrocycles in human blood can me about 2-24 h, 4-24 h, 6-24 h, 8-24 h, 10-24 h, 12-24 h, 14-24 h, 16-24 h, 18-24 h, 20-24 h, 22-24 h, 1-20 h, 4-20 h, 6-20 h, 8-20 h, 10-20 h, 12-20 h, 14-20 h, 16-20 h, 18-20 h, 1-16 h, 4-16 h, 6-16 h, 8-16 h, 10-16 h, 12-16 h, 14-16 h, 1-12 h, 4-12 h, 6-12 h, 8-12 h, 10-12 h, 1-8 h, 4-8 h, 6-8 h, or 1-4 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood can be bout 1-12 h, for example about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 2 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 4 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 6 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 8 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 10 h.

The half-life of the peptidomimetic macrocycles in biological tissue can be about 1-24 h. For example the circulating half-life of the peptidomimetic macrocycles in human blood can me about 1-24 h, 5-24 h, 10-24 h, 15-24 h, 20-24 h, 1-22 h, 5-22 h, 10-22 h, 15-22 h, 20-22 h, 1-20 h, 5-20 h, 15-20 h, 1-18 h, 5-18 h, 10-18 h, 15-18 h, 1-16 h, 5-16 h, 10-16 h, 15-16 h, 1-14 h, 5-14 h, 10-14 h, 1-12 h, 5-12 h, 10-12 h, 1-10 h, 5-10 h, 1-8 h, 5-8 h, 1-6 h, 5-6 h, or 1-4 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood can be bout 5-20 h, for example about 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h or 20 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 2 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 4 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 6 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 8 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 10 h.

The circulating half-life of the peptidomimetic macrocycles in human blood can be greater than, equal to, or less than the half-life of the peptidomimetic macrocycles in biological tissue. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood can be greater than the half-life of the peptidomimetic macrocycles in biological tissue. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood can be equal to the half-life of the peptidomimetic macrocycles in biological tissue. In some examples, the half-life of the peptidomimetic macrocycles in biological tissue is greater than the circulating half-life of the peptidomimetic macrocycles in human blood. This can facilitate administration of the peptidomimetic macrocycles at a lower dose and/or at lower frequency. In some embodiments, the half-life of the peptidomimetic macrocycles in biological tissue is at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 7 h, at least 8 h, at least 9 h, at least 10 h, at least 11 h, or at least 12 h greater than the than the circulating half-life of the peptidomimetic macrocycles in human blood. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 4 h and the half-life of the in biological tissue is about 10 h. In some examples, the circulating half-life of the peptidomimetic macrocycles in human blood is about 6 h and the half-life of the in biological tissue is about 10 h.

Preparation of Peptidomimetic Macrocycles

Peptidomimetic macrocycles can be prepared by any of a variety of methods known in the art. For example, any of the residues indicated by “$” or “$r8” in Table 3, Table 3a, Table 3b, or Table 3c can be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule or a precursor of such a residue.

Various methods to effect formation of peptidomimetic macrocycles are known in the art. For example, the preparation of peptidomimetic macrocycles of Formula I is described in Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. The α,α-disubstituted amino acids and amino acid precursors disclosed in the cited references can be employed in synthesis of the peptidomimetic macrocycle precursor polypeptides. For example, the “S5-olefin amino acid” is (S)-α-(2′-pentenyl) alanine and the “R8 olefin amino acid” is (R)-α-(2′-octenyl) alanine. Following incorporation of such amino acids into precursor polypeptides, the terminal olefins are reacted with a metathesis catalyst, leading to the formation of the peptidomimetic macrocycle. In various embodiments, the following amino acids can be employed in the synthesis of the peptidomimetic macrocycle:

In other embodiments, the peptidomimetic macrocycles are of Formula IV or IVa. Methods for the preparation of such macrocycles are described, for example, in U.S. Pat. No. 7,202,332.

Additional methods of forming peptidomimetic macrocycles which are envisioned as suitable include those disclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem (2003), 68, pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp. 1403-1406; U.S. Pat. Nos. 5,364,851; 5,446,128; 5,824,483; 6,713,280; and 7,202,332. In such embodiments, amino acid precursors are used containing an additional substituent R—at the alpha position. Such amino acids are incorporated into the macrocycle precursor at the desired positions, which can be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then effected according to the indicated method.

Assays

The properties of peptidomimetic macrocycles are assayed, for example, by using the methods described below. In some embodiments, a peptidomimetic macrocycle has improved biological properties relative to a corresponding polypeptide lacking the substituents described herein.

Assay to Determine α-Helicity

In solution, the secondary structure of polypeptides with α-helical domains will reach a dynamic equilibrium between random coil structures and α-helical structures, often expressed as a “percent helicity”. Thus, for example, alpha-helical domains are predominantly random coils in solution, with α-helical content usually under 25%. Peptidomimetic macrocycles with optimized linkers, on the other hand, possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide. In some embodiments, macrocycles will possess an alpha-helicity of greater than 50%. To assay the helicity of peptidomimetic macrocycles, the compounds are dissolved in an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7, or distilled H₂O, to concentrations of 25-50 μM). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710) using standard measurement parameters (e.g. temperature, 20° C.; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g. [Φ]222obs) by the reported value for a model helical decapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm)

A peptidomimetic macrocycle comprising a secondary structure such as an α-helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide. Typically peptidomimetic macrocycles exhibit Tm of >60° C. representing a highly stable structure in aqueous solutions. To assay the effect of macrocycle formation on melting temperature, peptidomimetic macrocycles or unmodified peptides are dissolved in distilled H₂O (e.g. at a final concentration of 50 μM) and the Tm is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710) using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1° C./min; path length, 0.1 cm).

Protease Resistance Assay

The amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, typically buries the amide backbone and therefore can shield it from proteolytic cleavage. The peptidomimetic macrocycles can be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide. For example, the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S/E ˜125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction displays first order kinetics and the rate constant, k, is determined from a plot of ln[S] versus time (k=−1Xslope).

Ex Vivo Stability Assay

Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more. For ex vivo serum stability studies, a variety of assays can be used. For example, a peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide (2 mcg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37° C. for 0, 1, 2, 4, 8, and 24 hours. To determine the level of intact compound, the following procedure can be used: The samples are extracted by transferring 100 μl of sera to 2 ml centrifuge tubes followed by the addition of 10 μL of 50% formic acid and 500 μL acetonitrile and centrifugation at 14,000 RPM for 10 min at 4±2° C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N₂<10 psi, 37° C. The samples are reconstituted in 100 μL of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis.

In Vitro Binding Assays

To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, a fluorescence polarization assay (FPA) is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25-1000 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values can be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic macrocycle shows, In some embodiments, similar or lower Kd than a corresponding uncrosslinked polypeptide.

In Vitro Displacement Assays to Characterize Antagonists of Peptide-Protein Interactions

To assess the binding and affinity of compounds that antagonize the interaction between a peptide and an acceptor protein, a fluorescence polarization assay (FPA) utilizing a fluoresceinated peptidomimetic macrocycle derived from a peptidomimetic precursor sequence is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution). A compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.

For example, putative antagonist compounds (1 nM to 1 mM) and a fluoresceinated peptidomimetic macrocycle (25 nM) are incubated with the acceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Antagonist binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values can be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.).

Any class of molecule, such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.

Assay for Protein-Ligand Binding by Affinity Selection-Mass Spectrometry

To assess the binding and affinity of test compounds for proteins, an affinity-selection mass spectrometry assay is used, for example. Protein-ligand binding experiments are conducted according to the following representative procedure outlined for a system-wide control experiment using 1 μM peptidomimetic macrocycle plus 5 μM hMDM2. A 1 μL DMSO aliquot of a 40 μM stock solution of peptidomimetic macrocycle is dissolved in 19 μL of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl). The resulting solution is mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To a 4 μL aliquot of the resulting supernatant is added 4 μL of 10 μM hMDM2 in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 Gg) of protein at 5.0 μM concentration in PBS plus 1 μM peptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated for 60 min at room temperature, and then chilled to 4° C. prior to size-exclusion chromatography-LC-MS analysis of 5.0 μL injections. Samples containing a target protein, protein-ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from non-binding component by a rapid SEC step. The SEC column eluate is monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, is well resolved from unbound components that are retained on the column. After the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it enters a sample loop where it is excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism. The (M+3H)³⁻ion of the peptidomimetic macrocycle is observed by ESI-MS at the expected m/z, confirming the detection of the protein-ligand complex.

Assay for Protein-Ligand Kd Titration Experiments

To assess the binding and affinity of test compounds for proteins, a protein-ligand Kd titration experiment is performed, for example. Protein-ligand K_(d) titrations experiments are conducted as follows: 2 μL DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (5, 2.5, . . . , 0.098 mM) are prepared then dissolved in 38 μL of PBS. The resulting solutions are mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2 in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 Gg) of protein at 5.0 μM concentration in PBS, varying concentrations (125, 62.5, . . . , 0.24 μM) of the titrant peptide, and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 30 min, then chilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections. The (M+H)¹⁺, (M+2H)²⁺, (M+3H)³⁺, and/or (M+Na)¹⁺ ion is observed by ESI-MS; extracted ion chromatograms are quantified, then fit to equations to derive the binding affinity K_(d) as described in “A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures.” Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization ofProtein-Ligand Interactions” D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Höfner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.

Assay for Competitive Binding Experiments by Affinity Selection-Mass Spectrometry

To determine the ability of test compounds to bind competitively to proteins, an affinity selection mass spectrometry assay is performed, for example. A mixture of ligands at 40 μM per component is prepared by combining 2 μL aliquots of 400 μM stocks of each of the three compounds with 14 μL of DMSO. Then, 1 μL aliquots of this 40 μM per component mixture are combined with 1 μL DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . , 0.078 mM). These 2 μL samples are dissolved in 38 μL of PBS. The resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10,000 g for 10 min. To 4.0 μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2 protein in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μM concentration in PBS plus 0.5 μM ligand, 2.5% DMSO, and varying concentrations (125, 62.5, . . . , 0.98 μM) of the titrant peptidomimetic macrocycle. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 60 min, then chilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections. Additional details on these and other methods are provided in “A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures.” Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J Am. Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions” D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Höfner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.

Binding Assays in Intact Cells

It is possible to measure binding of peptides or peptidomimetic macrocycles to their natural acceptors in intact cells by immunoprecipitation experiments. For example, intact cells are incubated with fluoresceinated (FITC-labeled) compounds for 4 hrs in the absence of serum, followed by serum replacement and further incubation that ranges from 4-18 hrs. Cells are then pelleted and incubated in lysis buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed by further 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of 50% bead slurry). After quick centrifugation, the pellets are washed in lysis buffer containing increasing salt concentration (e.g., 150, 300, 500 mM). The beads are then re-equilibrated at 150 mM NaCl before addition of SDS-containing sample buffer and boiling. After centrifugation, the supernatants are optionally electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots are optionally incubated with an antibody that detects FITC and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle.

Cellular Penetrability Assays

A peptidomimetic macrocycle is, for example, more cell penetrable compared to a corresponding uncrosslinked macrocycle. Peptidomimetic macrocycles with optimized linkers possess, for example, cell penetrability that is at least two-fold greater than a corresponding uncrosslinked macrocycle, and often 20% or more of the applied peptidomimetic macrocycle will be observed to have penetrated the cell after 4 hours. To measure the cell penetrability of peptidomimetic macrocycles and corresponding uncrosslinked macrocycle, intact cells are incubated with fluorescently-labeled (e.g. fluoresceinated) peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10 μM) for 4 hrs in serum free media at 37° C., washed twice with media and incubated with trypsin (0.25%) for 10 min at 37° C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan® HCS Reader.

Cellular Efficacy Assays

The efficacy of certain peptidomimetic macrocycles is determined, for example, in cell-based killing assays using a variety of tumorigenic and non-tumorigenic cell lines and primary cells derived from human or mouse cell populations. Cell viability is monitored, for example, over 24-96 hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) to identify those that kill at EC₅₀<10 μM. Several standard assays that measure cell viability are commercially available and are optionally used to assess the efficacy of the peptidomimetic macrocycles. In addition, assays that measure Annexin V and caspase activation are optionally used to assess whether the peptidomimetic macrocycles kill cells by activating the apoptotic machinery. For example, the Cell Titer-glo assay is used which determines cell viability as a function of intracellular ATP concentration.

In Vivo Stability Assay

To investigate the in vivo stability of the peptidomimetic macrocycles, the compounds are, for example, administered to mice and/or rats by IV, IP, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8 hrs and 24 hours post-injection. Levels of intact compound in 25 μL of fresh serum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models

To determine the anti-oncogenic activity of peptidomimetic macrocycles in vivo, the compounds are, for example, given alone (IP, IV, PO, by inhalation or nasal routes) or in combination with sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide). In one example, 5×10⁶ RS4;11 cells (established from the bone marrow of a patient with acute lymphoblastic leukemia) that stably express luciferase are injected by tail vein in NOD-SCID mice 3 hrs after they have been subjected to total body irradiation. If left untreated, this form of leukemia is fatal in 3 weeks in this model. The leukemia is readily monitored, for example, by injecting the mice with D-luciferin (60 mg/kg) and imaging the anesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton, Mass.). Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software (Caliper Life Sciences, Hopkinton, Mass.). Peptidomimetic macrocycles alone or in combination with sub-optimal doses of relevant chemotherapeutics agents are, for example, administered to leukemic mice (10 days after injection/day 1 of experiment, in bioluminescence range of 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughout the experiment every other day and survival monitored daily for the duration of the experiment. Expired mice are optionally subjected to necropsy at the end of the experiment. Another animal model is implantation into NOD-SCID mice of DoHH2, a cell line derived from human follicular lymphoma, that stably expresses luciferase. These in vivo tests optionally generate preliminary pharmacokinetic, pharmacodynamic and toxicology data.

Clinical Trials

To determine the suitability of the peptidomimetic macrocycles for treatment of humans, clinical trials are performed. For example, patients diagnosed with liquid cancer and in need of treatment can be selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle, while the control groups receive a placebo or a known anti-cancer drug. The treatment safety and efficacy of the peptidomimetic macrocycles can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life. In this example, the patient group treated with a peptidomimetic macrocycle can show improved long-term survival compared to a patient control group treated with a placebo.

Formulation and Administration Mode of Administration

An effective amount of a peptidomimetic macrocycles of the disclosure can be administered in either single or multiple doses by any of the accepted modes of administration. In some embodiments, the peptidomimetic macrocycles of the disclosure are administered parenterally, for example, by subcutaneous, intramuscular, intrathecal, intravenous or epidural injection. For example, the peptidomimetic macrocycle is administered intravenously, intraarterially, subcutaneously or by infusion. In some examples, the peptidomimetic macrocycle is administered intravenously. In some examples, the peptidomimetic macrocycle is administered intraarterially.

Regardless of the route of administration selected, the peptidomimetic macrocycles of the present disclosure, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutically-acceptable dosage forms. The peptidomimetic macrocycles according to the disclosure can be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

In one aspect, the disclosure provides pharmaceutical formulation comprising a therapeutically-effective amount of one or more of the peptidomimetic macrocycles described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. In one embodiment, one or more of the peptidomimetic macrocycles described herein are formulated for parenteral administration for parenteral administration, one or more peptidomimetic macrocycles disclosed herein can be formulated as aqueous or nonaqueous solutions, dispersions, suspensionsor emulsions or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use. Such formulations can comprise sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. If desired the formulation can be diluted prior to use with, for example, an isotonic saline solution or a dextrose solution. In some examples, the peptidomimetic macrocycle is formulated as an aqueous solution and is administered intravenously.

Amount and Frequency of Administration

Dosing can be determined using various techniques. The selected dosage level can depend upon a variety of factors including the activity of the particular peptidomimetic macrocycle employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular peptidomimetic macrocycle being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular peptidomimetic macrocycle employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. The dosage values can also vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

A physician or veterinarian can prescribe the effective amount of the compound required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In some embodiments, a suitable daily dose of a peptidomimetic macrocycle of the disclosure can be that amount of the peptidomimetic macrocycle which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. The precise time of administration and amount of any particular peptidomimetic macrocycle that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular peptidomimetic macrocycle, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like.

Dosage can be based on the amount of the peptidomimetic macrocycle per kg body weight of the patient. Alternatively, the dosage of the subject disclosure can be determined by reference to the plasma concentrations of the peptidomimetic macrocycle. For example, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity (AUC) can be used.

In some embodiments, the subject is a human subject and the amount of the compound administered is 0.01-100 mg per kilogram body weight of the human subject. For example, in various examples, the amount of the compound administered is about 0.01-50 mg/kg, about 0.01-20 mg/kg, about 0.01-10 mg/kg, about 0.1-100 mg/kg, about 0.1-50 mg/kg, about 0.1-20 mg/kg, about 0.1-10 mg/kg, about 0.5-100 mg/kg, about 0.5-50 mg/kg, about 0.5-20 mg/kg, about 0.5-10 mg/kg, about 1-100 mg/kg, about 1-50 mg/kg, about 1-20 mg/kg, about 1-10 mg/kg body weight of the human subject. In one embodiment, about 0.5 mg-10 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered. In some examples the amount of the compound administered is about 0.16 mg, about 0.32 mg, about 0.64 mg, about 1.28 mg, about 3.56 mg, about 7.12 mg, about 14.24 mg, or about 20 mg per kilogram body weight of the human subject. In some examples the amount of the compound administered is about 0.16 mg, about 0.32 mg, about 0.64 mg, about 1.28 mg, about 3.56 mg, about 7.12 mg, or about 14.24 mg per kilogram body weight of the human subject. In some examples the amount of the compound administered is about 0.16 mg per kilogram body weight of the human subject. In some examples the amount of the compound administered is about 0.32 mg per kilogram body weight of the human subject. In some examples the amount of the compound administered is about 0.64 mg per kilogram body weight of the human subject. In some examples the amount of the compound administered is about 1.28 mg per kilogram body weight of the human subject. In some examples the amount of the compound administered is about 3.56 mg per kilogram body weight of the human subject. In some examples the amount of the compound administered is about 7.12 mg per kilogram body weight of the human subject. In some examples the amount of the compound administered is about 14.24 mg per kilogram body weight of the human subject.

In some embodiments about 0.5- about 20 mg or about 0.5- about 10 mg of the compound per kilogram body weight of the human subject is administered two times a week. For example about 0.5- about 1 mg, about 0.5- about 5 mg, about 0.5- about 10 mg, about 0.5- about 15 mg, about 1- about 5 mg, about 1- about 10 mg, about 1- about 15 mg, about 1- about 20 mg, about 5- about 10 mg, about 1- about 15 mg, about 5- about 20 mg, about 10- about 15 mg, about 10- about 20 mg, or about 15- about 20 mg of the compound per kilogram body weight of the human subject is administered about twice a week. In some examples about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, about 5 mg, about 5.25 mg, about 5.5 mg, about 5.75 mg, about 6 mg, about 6.25 mg, about 6.5 mg, about 6.75 mg, about 7 mg, about 7.25 mg, about 7.5 mg, about 7.75 mg, about 8 mg, about 8.25 mg, about 8.5 mg, about 8.75 mg, about 9 mg, about 9.25 mg, about 9.5 mg, about 9.75 mg, about 10 mg, about 10.25 mg, about 10.5 mg, about 10.75 mg, about 11 mg, about 11.25 mg, about 11.5 mg, about 11.75 mg, about 12 mg, about 12.25 mg, about 12.5 mg, about 12.75 mg, about 13 mg, about 13.25 mg, about 13.5 mg, about 13.75 mg, about 14 mg, about 14.25 mg, about 14.5 mg, about 14.75 mg, about 15 mg, about 15.25 mg, about 15.5 mg, about 15.75 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20 mg of the compound per kilogram body weight of the human subject is administered two times a week. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered two times a week. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg or about 10 mg per kilogram body weight of the human subject and the compound is administered two times a week.

In some embodiments about 0.5- about 20 mg or about 0.5- about 10 mg of the compound per kilogram body weight of the human subject is administered once a week. For example about 0.5- about 1 mg, about 0.5- about 5 mg, about 0.5- about 10 mg, about 0.5- about 15 mg, about 1- about 5 mg, about 1- about 10 mg, about 1- about 15 mg, about 1- about 20 mg, about 5- about 10 mg, about 1- about 15 mg, about 5- about 20 mg, about 10- about 15 mg, about 10- about 20 mg, or about 15- about 20 mg of the compound per kilogram body weight of the human subject is administered once a week. In some examples about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, about 5 mg, about 5.25 mg, about 5.5 mg, about 5.75 mg, about 6 mg, about 6.25 mg, about 6.5 mg, about 6.75 mg, about 7 mg, about 7.25 mg, about 7.5 mg, about 7.75 mg, about 8 mg, about 8.25 mg, about 8.5 mg, about 8.75 mg, about 9 mg, about 9.25 mg, about 9.5 mg, about 9.75 mg, about 10 mg, about 10.25 mg, about 10.5 mg, about 10.75 mg, about 11 mg, about 11.25 mg, about 11.5 mg, about 11.75 mg, about 12 mg, about 12.25 mg, about 12.5 mg, about 12.75 mg, about 13 mg, about 13.25 mg, about 13.5 mg, about 13.75 mg, about 14 mg, about 14.25 mg, about 14.5 mg, about 14.75 mg, about 15 mg, about 15.25 mg, about 15.5 mg, about 15.75 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20 mg of the compound per kilogram body weight of the human subject is administered once a week. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered once a week. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg or about 10 mg per kilogram body weight of the human subject and the compound is administered once a week

In some embodiments about 0.5- about 20 mg or about 0.5- about 10 mg of the compound per kilogram body weight of the human subject is administered 3, 4, 5, 6, or 7 times a week. For example, about 0.5- about 1 mg, about 0.5- about 5 mg, about 0.5- about 10 mg, about 0.5- about 15 mg, about 1- about 5 mg, about 1- about 10 mg, about 1- about 15 mg, about 1- about 20 mg, about 5- about 10 mg, about 1- about 15 mg, about 5- about 20 mg, about 10- about 15 mg, about 10- about 20 mg, or about 15- about 20 mg of the compound per kilogram body weight of the human subject is administered 3, 4, 5, 6, or 7 times a week. In some examples about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, about 5 mg, about 5.25 mg, about 5.5 mg, about 5.75 mg, about 6 mg, about 6.25 mg, about 6.5 mg, about 6.75 mg, about 7 mg, about 7.25 mg, about 7.5 mg, about 7.75 mg, about 8 mg, about 8.25 mg, about 8.5 mg, about 8.75 mg, about 9 mg, about 9.25 mg, about 9.5 mg, about 9.75 mg, about 10 mg, about 10.25 mg, about 10.5 mg, about 10.75 mg, about 11 mg, about 11.25 mg, about 11.5 mg, about 11.75 mg, about 12 mg, about 12.25 mg, about 12.5 mg, about 12.75 mg, about 13 mg, about 13.25 mg, about 13.5 mg, about 13.75 mg, about 14 mg, about 14.25 mg, about 14.5 mg, about 14.75 mg, about 15 mg, about 15.25 mg, about 15.5 mg, about 15.75 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20 mg of the compound per kilogram body weight of the human subject is administered 3, 4, 5, 6, or 7 times a week. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered 3, 4, 5, 6, or 7 times a week. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg, or about 10 mg per kilogram body weight of the human subject and the compound is administered 3, 4, 5, 6, or 7 times a week.

In some embodiments, about 0.5- about 20 mg or about 0.5- about 10 mg of the compound per kilogram body weight of the human subject is administered once every 2, 3, or 4 weeks. For example, about 0.5- about 1 mg, about 0.5- about 5 mg, about 0.5- about 10 mg, about 0.5- about 15 mg, about 1- about 5 mg, about 1- about 10 mg, about 1- about 15 mg, about 1- about 20 mg, about 5- about 10 mg, about 1- about 15 mg, about 5- about 20 mg, about 10- about 15 mg, about 10- about 20 mg, or about 15- about 20 mg of the compound per kilogram body weight of the human subject is administrated 3, 4, 5, 6, or 7 once every 2 or 3 week. In some examples about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, about 5 mg, about 5.25 mg, about 5.5 mg, about 5.75 mg, about 6 mg, about 6.25 mg, about 6.5 mg, about 6.75 mg, about 7 mg, about 7.25 mg, about 7.5 mg, about 7.75 mg, about 8 mg, about 8.25 mg, about 8.5 mg, about 8.75 mg, about 9 mg, about 9.25 mg, about 9.5 mg, about 9.75 mg, about 10 mg, about 10.25 mg, about 10.5 mg, about 10.75 mg, about 11 mg, about 11.25 mg, about 11.5 mg, about 11.75 mg, about 12 mg, about 12.25 mg, about 12.5 mg, about 12.75 mg, about 13 mg, about 13.25 mg, about 13.5 mg, about 13.75 mg, about 14 mg, about 14.25 mg, about 14.5 mg, about 14.75 mg, about 15 mg, about 15.25 mg, about 15.5 mg, about 15.75 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20 mg of the compound per kilogram body weight of the human subject is administered once every 2 or 3 weeks. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered once every 2 weeks. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg or about 10 mg per kilogram body weight of the human subject and the compound is administered once every 2 weeks. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered once every 3 weeks. In some examples, the amount of the compound administered is about 1.25 mg, about 2.5 mg, about 5 mg, or about 10 mg per kilogram body weight of the human subject and the compound is administered once every 3 weeks.

In some embodiments, the compound is administered gradually over a period of time. A desired amount of compound can, for example can be administered gradually over a period of from about 0.1 h-24 h. In some cases a desired amount of compound is administered gradually over a period of 0.1 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h. In some examples, a desired amount of compound is administered gradually over a period of 0.25-12 h, for example over a period of 0.25-1 h, 0.25-2 h, 0.25-3 h, 0.25-4 h, 0.25-6 h, 0.25-8 h, 0.25-10 h. In some examples, a desired amount of compound is administered gradually over a period of 0.25-2 h. In some examples, a desired amount of compound is administered gradually over a period of 0.25-1 h. In some examples, a desired amount of compound is administered gradually over a period of 0.25 h, 0.3 h, 0.4 h, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, or 2.0 h. In some examples, a desired amount of compound is administered gradually over a period of 1 h. In some examples, a desired amount of compound is administered gradually over a period of 2 h.

Administration of the compound can continue as long as necessary. In some embodiments, one or more compound of the disclosure is administered for more than 1 day, more than 1 week, more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, more than 12 months, more than 13 months, more than 14 months, more than 15 months, more than 16 months, more than 17 months, more than 18 months, more than 19 months, more than 20 months, more than 21 months, more than 22 months, more than 23 months, or more than 24 months. In some embodiments, one or more compound of the disclosure is administered for less than 1 week, less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, less than 9 months, less than 10 months, less than 11 months, less than 12 months, less than 13 months, less than 14 months, less than 15 months, less than 16 months, less than 17 months, less than 18 months, less than 19 months, less than 20 months, less than 21 months, less than 22 months, less than 23 months, or less than 24 months.

In some embodiments, the compound is administered on day 1, 8, 15 and 28 of a 28 day cycle. In some embodiments, the compound is administered on day 1, 8, 15 and 28 of a 28 day cycle and administration is continued for two cycles. In some embodiments, the compound is administered on day 1, 8, 15 and 28 of a 28 day cycle and administration is continued for three cycles. In some embodiments, the compound is administered on day 1, 8, 15 and 28 of a 28 day cycle and administration is continued for 4, 5, 6, 7, 8, 9, 10, or more cycles.

In some embodiments, the compound is administered on day 1, 8, 11 and 21 of a 21 day cycle. In some embodiments, the compound is administered on day 1, 8, 11 and 21 of a 21 day cycle and administration is continued for two cycles. In some embodiments, the compound is administered on day 1, 8, 11 and 21 of a 21 day cycle and administration is continued for three cycles. In some embodiments, the compound is administered on day 1, 8, 11 and 21 of a 21 day cycle and administration is continued for 4, 5, 6, 7, 8, 9, 10, or more cycles.

In some embodiments, one or more compound of the disclosure is administered chronically on an ongoing basis. In some embodiments administration of one or more compound of the disclosure is continued until documentation of disease progression, unacceptable toxicity, or patient or physician decision to discontinue administration.

Method and Uses

In one aspect, the disclosure provides a method of treating liquid cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In some embodiments, the peptidomimetic macrocycle can disrupt the interaction between p53 and MDM2 and MDMX. In some embodiments, treatment according to the method disclosed herein can result in re-activation of the p53 pathway, decreased liquid cancer cell proliferation, increased p53 protein, increased p21, and/or increased apoptosis in the human subject.

In one aspect, the disclosure provides a method of treating liquid cancer, determined to lack a p53 deactivating mutation, in a subject the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In some embodiments, the peptidomimetic macrocycle can disrupt the interaction between p53 and MDM2 and MDMX. The method further can comprise confirming the lack of the p53 deactivating mutation in the subject prior to the administration of the peptidomimetic macrocycle. In some embodiments, treatment according to the method disclosed herein can result in re-activation of the p53 pathway, decreased liquid cancer cell proliferation, increased p53 protein, increased p21, and/or increased apoptosis in the human subject.

In one aspect, the disclosure provides a method of treating liquid cancer in a subject expressing wild type p53, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In some embodiments, the peptidomimetic macrocycle can disrupt the interaction between p53 and MDM2 and MDMX. The method further can comprise confirming the wild type p53 status of the subject prior to the administration of the peptidomimetic macrocycle. In some embodiments, treatment according to the method disclosed herein can result in re-activation of the p53 pathway, decreased liquid cancer cell proliferation, increased p53 protein, increased p21, and/or increased apoptosis in the human subject.

In some embodiments, the methods for treating liquid cancer provided herein inhibit, reduce, diminish, arrest, or stabilize a liquid cancer cell, such as a CTC or an MNBC, associated with the liquid cancer. In other embodiments, the methods for treating liquid cancer provided herein inhibit, reduce, diminish, arrest, or stabilize the symptoms associated with the liquid cancer or two or more symptoms thereof. In some examples, the methods for treating liquid cancer provided herein cause the reduction in the number of liquid cancer cells and/or one or more symptoms associated with the liquid cancer. In other examples, the methods for treating liquid cancer provided herein maintain the number of liquid cancer cells so that they donot increase, or so that the number of liquid cancer cells increases by less than the increase of a number of liquid cancer cells after administration of a standard therapy as measured by, for example, conventional methods available to one of skill in the art, such as ultrasound, CT Scan, MRI, dynamic contrast-enhanced MRI, or PET Scan. In some examples, the methods for treating liquid cancer provided herein decrease the number of liquid cancer cells. In some examples, the methods for treating liquid cancer provided herein reduce the formation of liquid cancer cells. In some examples, the methods for treating liquid cancer provided herein eradicate, remove, or control primary, regional and/or metastatic liquid cancer cells associated with the liquid cancer. In some examples, the methods for treating liquid cancer provided herein decrease the number or size of metastases associated with the liquid cancer. In some examples, the methods for treating liquid cancer provided herein reduce the number of liquid cancer cells in a subject by an amount in the range of about 5- about 10%, about 5- about 20%, about 10- about 20%, about 15- about 20%, about 10- about 30%, about 20- about 30%, about 20- about 40%, about 30- about 40%, about 10- about 50%, about 20- about 50%, about 30- about 50%, about 40- about 50%, about 10- about 60%, about 20- about 60%, about 30- about 60%, about 40- about 60%, about 50- about 60%, about 10- about 70%, about 20- about 70%, about 30- about 70%, about 40- about 70%, about 50- about 70%, about 60- about 70%, about 10- about 80%, about 20- about 80%, about 30- about 80%, about 40- about 80%, about 50- about 80%, about 60- about 80%, about 70- about 80%, about 10- about 90%, about 20- about 90%, about 30- about 90%, about 40- about 90%, about 50- about 90%, about 60- about 90%, about 70- about 90%, about 80- about 90%, about 10- about 100%, about 20%- about 100%, about 30- about 100%, about 40- about 100%, about 50- about 100%, about 60- about 100%, about 70- about 100%, about 80- about 100%, about 90- about 100%, about 95- about 100%, or any range in between, relative to the number of liquid cancer cells in a subject prior to administration of the peptidomimetic macrocycles as assessed by, for example, CT Scan, MRI, DCE-MRI, or PET Scan. In certain embodiments, the methods herein reduce the number of liquid cancer cells in a subject by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100%, relative to the number of liquid cancer cells prior to administration of the peptidomimetic macrocycle as assessed by, for example, CT Scan, MRI, DCE-MRI, or PET Scan.

In some embodiments, the methods provided herein reduce the liquid cancer cell perfusion in a subject by an amount in the range of about 5- about 10%, about 5- about 20%, about 10- about 20%, about 15- about 20%, about 10- about 30%, about 20- about 30%, about 20- about 40%, about 30- about 40%, about 10- about 50%, about 20- about 50%, about 30- about 50%, about 40- about 50%, about 10- about 60%, about 20- about 60%, about 30- about 60%, about 40- about 60%, about 50- about 60%, about 10- about 70%, about 20- about 70%, about 30- about 70%, about 40- about 70%, about 50- about 70%, about 60- about 70%, about 10- about 80%, about 20- about 80%, about 30- about 80%, about 40- about 80%, about 50- about 80%, about 60- about 80%, about 70- about 80%, about 10- about 90%, about 20- about 90%, about 30- about 90%, about 40- about 90%, about 50- about 90%, about 60- about 90%, about 70- about 90%, about 80- about 90%, about 10- about 100%, about 20%- about 100%, about 30- about 100%, about 40- about 100%, about 50- about 100%, about 60- about 100%, about 70- about 100%, about 80- about 100%, about 90- about 100%, about 95- about 100%, or any range in between, relative to liquid cancer cell perfusion prior to administration of the peptidomimetic macrocycle, as assessed by, for example, MRI, DCE-MRI, or PET Scan. In certain embodiments, the methods provided herein reduce the liquid cancer cell perfusion in a subject by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%, relative to liquid cancer cell perfusion prior to administration of the peptidomimetic macrocycle as assessed by, for example, MRI, DCE-MRI, or PET Scan.

In some embodiments, the methods provided herein inhibit or decrease liquid cancer cell metabolism in a subject in the range of about 5- about 10%, about 5- about 20%, about 10- about 20%, about 15- about 20%, about 10- about 30%, about 20- about 30%, about 20- about 40%, about 30- about 40%, about 10- about 50%, about 20- about 50%, about 30- about 50%, about 40- about 50%, about 10- about 60%, about 20- about 60%, about 30- about 60%, about 40- about 60%, about 50- about 60%, about 10- about 70%, about 20- about 70%, about 30- about 70%, about 40- about 70%, about 50- about 70%, about 60- about 70%, about 10- about 80%, about 20- about 80%, about 30- about 80%, about 40- about 80%, about 50- about 80%, about 60- about 80%, about 70- about 80%, about 10- about 90%, about 20- about 90%, about 30- about 90%, about 40- about 90%, about 50- about 90%, about 60- about 90%, about 70- about 90%, about 80- about 90%, about 10- about 100%, about 20%- about 100%, about 30- about 100%, about 40- about 100%, about 50- about 100%, about 60- about 100%, about 70- about 100%, about 80- about 100%, about 90- about 100%, about 95- about 100%, or any range in between, relative to liquid cancer cell metabolism prior to administration of the peptidomimetic macrocycle, as assessed by, for example, MRI, DCE-MRI, or PET Scan. In certain embodiments, the methods provided herein inhibit or decrease liquid cancer cell metabolism in a subject as assessed by, for example,PET scanning. In specific embodiments, the methods provided herein inhibit or decrease liquid cancer cell metabolism in a subject by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, relative to liquid cancer cell metabolism prior to administration of the peptidomimetic macrocycle.

In other aspect, the disclosure provides a method for increasing the survival time of a subject with liquid cancer determined to lack a p53 deactivating mutation and/or with liquid cancer expressing wild type p53, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In some examples, the survival time of the subject is at least 30 days longer than the expected survival time of the subject if the subject was not treated according to the methods provided herein. In some examples, the survival time of the subject is at 1 month- about 5 years longer than the expected survival time of the subject if the subject was not treated according to the methods provided herein. For example, the survival time of the subject is at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, or at least 24 months longer than the expected survival time of the subject if the subject was not treated according to the methods disclosed herein disclosure.

In one aspect, the disclosure provides a method to assessed presence, absence or amount of the biomarker in a subject suffering with liquid cancer. In some examples, the biomarkers include patient biomarkers, for example, the p53 status of the subject and the MDM2 and MDMX expression levels in the subject.

The method of the disclosure can also optionally include studying and/or evaluating the safety and/or tolerability of the peptidomimetic macrocycles disclosed herein in the subject.

Also provided herein is a method to re-activate the p53 pathway in a subject with a liquid cancer lacking a p53 deactivating mutation and/or expressing wild type p53, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

Also provided herein is a method to decrease liquid cancer cell proliferation in a human subject with a liquid cancer lacking a p53 deactivating mutation and/or expressing wild type p53, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

Also provided herein is a method to increased p53 protein in a subject with a liquid cancer lacking a p53 deactivating mutation and/or expressing wild type p53, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

Also provided herein is a method to increased p21 in a subject with a liquid cancer lacking a p53 deactivating mutation and/or expressing wild type p53, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

Also provided herein is a method to increased apoptosis in a subject with a liquid cancer lacking a p53 deactivating mutation and/or expressing wild type p53, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.

In some embodiments, the disclosure also provides a method to determine the dose limiting toxicities (DLTs) and/or maximum tolerated dose (MTD or OBD) or the optimal biological dose (OBD) of the peptidomimetic macrocycles disclosed herein in subject with a liquid cancer (e.g., a liquid lymphoma) lacking a p53 deactivating mutation and/or expressing wild type p53.

The methods of the disclosure can optionally include pharmacokinetic analysis of the peptidomimetic macrocycles disclosed herein. Accordingly, the methods can further comprise collecting one or more biological sample from the subject at one or more specific time point and analyzing the one or more biological sample for levels of the peptidomimetic macrocycles and/or it metabolites. The biological sample can be a blood sample from the subject, for example, a blood sample from a human subject. The one or more specific time point can include time points before, after and/or during the administration of the peptidomimetic macrocycle to the subject. In some embodiments one or more biological sample includes biological samples collected before and after each administration of the peptidomimetic macrocycle to the subject. In some embodiments a biological sample for pharmacokinetic analysis is collected before the first administration of the peptidomimetic macrocycle to the subject and at one or more time points after each administration of the peptidomimetic macrocycles to the subject. The biological sample collected before the administration of the peptidomimetic macrocycle to the subject can be done within 0-24 hour before the start of administration of the peptidomimetic macrocycle to the subject. For example, the biological sample can be collected within 24 h, within 23 h, within 22 h, within 21 h, within 20 h, within 19 h, within 18 h, within 17 h, within 16 h, within 15 h, within 14 h, within 13 h, within 12 h, within 11 h, within 10 h, within 9 h, within 8 h, within 7 h, within 6 h, within 5 h, within 4 h, within 3 h, within 2 h, within 1 h, within 30 min, within 15 min, or immediately before the administration of the peptidomimetic macrocycle to the subject. One or more biological samples collected after the administration of the peptidomimetic macrocycle to the subject can be collected, for example after 0 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min, 1.25 h, 1.5 h, 1.75 h, 2.0 h, 2.25 h, 2.5 h, 2.75 h, 3.0 h, 3.25 h, 3.5 h, 3.75 h, 4.0 h, 4.25 h, 4.5 h, 4.75 h, 5.0 h, 5.25 h, 5.5 h, 5.75 h, 6.0 h, 6.25 h, 6.5 h, 6.75 h, 7.0 h, 7.25 h, 7.5 h, 7.75 h, 8.0 h, 8.25 h, 8.5 h, 8.75 h, 9.0 h, 9.25 h, 9.5 h, 9.75 h, 10.0 h, 10.25 h, 10.5 h, 10.75 h, 11.0 h, 11.25 h, 11.5 h, 11.75 h, 12.0 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, 52 h, 56 h, 60 h, 64 h, 68 h, 72 h, or 0-72 h after the administration of the peptidomimetic macrocycle to the subject. In some embodiments, the peptidomimetic macrocycle is administered on day 1, day 8, day 15 of a 28 day cycle and biological sample is collected before administration on day 1, after the administration on day 1 (multiple biological samples can be collected, for example after about 0 min, about 30 min, about 1 h, about 2 h, about 4 h, about 8 h, about 24 h, and 48 hour after administration), before administration on day 8, after administration on day 8 (multiple biological samples can be collected, for example after about 0 min, about 30 min, about 1 h, about 2 h, and about 4 h after administration), before administration on day 15 and after administration on day 15 (multiple biological samples can be collected, for example after about 0 min, about 30 min, about 1 h, about 2 h, about 4 h, about 8 h, and about 24 h after administration). In some embodiments, the peptidomimetic macrocycle is administered on day 1, day 8, day 11 of a 21 day cycle and biological sample is collected before administration on day 1, after the administration on day 1 (multiple biological samples can be collected, for example after about 0 min, about 30 min, about 1 h, about 2 h, about 4 h, about 8 h, about 24 h, and 48 hour after administration), before administration on day 8, after administration on day 8 (multiple biological samples can be collected, for example after about 0 min, about 30 min, about 1 h, about 2 h, and about 4 h after administration), before administration on day 11 and after administration on day 11 (multiple biological samples can be collected, for example after about 0 min, about 30 min, about 1 h, about 2 h, about 4 h, about 8 h, and about 24 h after administration).

The method of the disclosure can optionally include pharmacodynamic analysis of the peptidomimetic macrocycles disclosed herein. Accordingly, the methods can comprise collecting one or more biological samples from the subject at one or more specific time points for pharmacodynamic analysis. Pharmacodynamic analysis can include analyzing the levels of biomarkers including MIC-1, p53, MDM2, MDMX, p21 and/or cases in the biological sample. Detection of biomarkers in a biological sample can be performed by, for example, direct measurement, immunohistochemistry, immunoblotting, immunoflourescense, immunoabsorbence, immunoprecipitations, protein array, flourescence in situ hybridization, FACS analysis, hybridization, in situ hybridization, Northern blots, Southern blots, Western blots, ELISA, radioimmunoassay, gene array/chip, PCR, RT-PCR, or cytogenetic analysis. The biological sample for pharmacodynamic analysis can be a blood sample or a liquid cancer cell specimen from the subject, for example, a biological sample for pharmacodynamic analysis can be a blood sample or a liquid cancer cell specimen from the human subject. The biological samples for pharmacodynamic analysis of the peptidomimetic macrocycles can be collected any time before, during, or after the administration of the peptidomimetic macrocycle to the subject. In some embodiments a blood sample for pharmacokinetic analysis is collected before the first administration of the peptidomimetic macrocycle to the subject and at one or more time points after each administration of the peptidomimetic macrocycles to the subject. The blood sample collected before the administration of the peptidomimetic macrocycle to the subject can be done within 0-24 hour before the start of administration of the peptidomimetic macrocycle to the subject. For example, the biological sample can be collected within 24 h, within 23 h, within 22 h, within 21 h, within 20 h, within 19 h, within 18 h, within 17 h, within 16 h, within 15 h, within 14 h, within 13 h, within 12 h, within 11 h, within 10 h, within 9 h, within 8 h, within 7 h, within 6 h, within 5 h, within 4 h, within 3 h, within 2 h, within 1 h, within 30 min, within 15 min of, or immediately before the administration of the peptidomimetic macrocycle to the subject. One or more blood samples for pharmacodynamic analysis collected after the administration of the peptidomimetic macrocycle to the subject can be collected from 0- about 72 h, for example after about 12 h, after about 24 h, after about 36 h or after about 48 h after the administration of the peptidomimetic macrocycle to the subject. In some embodiments, the peptidomimetic macrocycle is administered on day 1, day 8, day 15 of a 28 day cycle and blood samples for pharmacodynamic analysis are collected before administration on day 1, after the administration on day 1 (multiple samples can be collected, for example after about 24 h and 48 hour after administration), before administration on day 8, after administration on day 8 (multiple samples can be collected, for example with about 1 h administration), before administration on day 15 and after administration on day 15 (multiple samples can be collected, for example with about 1 h and about 48 h after administration), and day 22. Biological samples for pharmacodynamic analysis can be collected at any time before, after or during the administration of the peptidomimetic macrocycle to the subject. For example the peptidomimetic macrocycle can be administered on day 1, day 8, day 15 of a 28 day cycle and liquid cancer cell samples for pharmacodynamic analysis are collected before administration on day 1 and between day 14-day 18, for example of day 16. In some embodiments, the peptidomimetic macrocycle is administered on day 1, day 8, day 11, of a 21 day cycle and blood samples for pharmacodynamic analysis are collected before administration on day 1, after the administration on day 1 (multiple samples can be collected, for example after about 24 h and 48 hour after administration), before administration on day 8, after administration on day 8 (multiple samples can be collected, for example with about 1 h administration), before administration on day 11 and after administration on day 11 (multiple samples can be collected, for example with about 1 h and about 48 h after administration), and day 22. Biological samples for pharmacodynamic analysis can be collected at any time before, after or during the administration of the peptidomimetic macrocycle to the subject. For example the peptidomimetic macrocycle can be administered on day 1, day 8, day 11 of a 21 day cycle and liquid cancer cell samples for pharmacodynamic analysis are collected before administration on day 1 and between day 10-day 14, for example of day 12.

The method of the disclosure can optionally include clinical activity analysis of the peptidomimetic macrocycles disclosed herein. Accordingly, the methods can comprise analyzing one or more biological samples collected from the subject at one or more specific time points. Any appropriate analytical procedure can be used for the analysis of the biological samples. For example, imaging techniques like radiographs, ultrasound, CT scan, PET scan, MRI scan, chest x-ray, laparoscopy, complete blood count (CBC) test, bone scanning and fecal occult blood test can be used. Further analytical procedures that can be used include blood chemistry analysis, chromosomal translocation analysis, needle biopsy, tissue biopsy, fluorescence in situ hybridization, laboratory biomarker analysis, immunohistochemistry staining method, flow cytometry, or a combination thereof. The method can further comprise tabulating and/or plotting results of the analytical procedure.

For example, pharmacodynamics can be assessed by laboratory-based evaluation of several biomarkers of p53 activation, including levels of p21, caspase and MDM2 in liquid cancer cell tissue, and where available in CTC, as well as MIC-1 in blood, before and after treatment with the peptidomimetic macrocycles.

Results available from previous genetic and biomarker tests, and additional tests of the blood and t liquid cancer cell samples for biomarkers relevant to the safety and efficacy of the peptidomimetic macrocycles can be investigated for possible correlation with patient outcome.

For example, clinical activity or response can be evaluated by standard imaging assessments, such as computed tomography (CT), magnetic resonance imaging (MRI), and bone scans. In addition, [¹⁸F]-fluorodeoxyglucose and [¹⁸F]-fluorothymidine positron emission tomography (FDG-PET and FLT-PET, respectively), or other techniques considered clinically appropriate for the patient's specific disease type can be used. CT-imaging can be performed, for example, at the end of Cycle 2, and every 2 cycles (e.g., Cycles 4 and 6) thereafter for DR-A and after the last infusion in Cycle 3 and every 3 cycles (e.g., Cycles 6 and 9) thereafter in DR-B. Anti-liquid cancer cell activity can be assessed using IWG (2014) (Appendix H) criteria for patients with liquid lymphomas. Additionally, for patients with an FDG-avid liquid lymphoma, FDG-PET imaging can be performed at baseline and post-baseline as outlined in IWG 2014. FLT-PET imaging can be performed at baseline for patients with liquid cancer cell commonly showing sufficient uptake of FLT tracer, e.g., patients with liquid lymphoma. For example, DR-A assigned patients who demonstrate a standard uptake value (SUV) of ≥5 at baseline can have a repeat FLT image one day after their last infusion of study medication in Cycle 1, i.e., Day 16. For example, DR-B patients who demonstrate a standard uptake value (SUV) of 5 at baseline can have a repeat FLT image one day after their last infusion of study medication in Cycle 1, i.e., Day 12.

Biological Samples

As used in the present application, “biological sample” means any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus, and the like. Also included within the meaning of the term “biological sample” is an organ or tissue extract and culture fluid in which any cells or tissue preparation from a subject has been incubated. Biological samples also include liquid cancer cell samples or specimens. Liquid cancer cell sample can be a liquid cancer cell tissue sample. In some embodiments, the liquid cancer cell tissue sample can obtained from surgically excised tissue. Tissue samples and cellular samples can also be obtained without invasive surgery, for example by punctuating the chest wall or the abdominal wall or from masses of breast, thyroid or other sites with a fine needle and withdrawing cellular material (fine needle aspiration biopsy). In some embodiments, a biological sample is a bone marrow aspirate sample.

The biological samples obtained can be used in fresh, frozen, or fixed (e.g., paraffin-embedded) form, depending on the nature of the sample, the assay used, and the convenience of the practitioner. Although fresh, frozen and fixed materials are suitable for various RNA and protein assays, generally, fresh tissues can be preferred for ex vivo measurements of activity.

Fixed tissue samples can also be employed. Tissue obtained by biopsy is often fixed, usually by formalin, formaldehyde, or gluteraldehyde, for example, or by alcohol immersion. Fixed biological samples are often dehydrated and embedded in paraffin or other solid supports. See the reference Plenat et al., 2001, Ann. Pathol. 21:29-47. Non-embedded, fixed tissue, as well as fixed and embedded tissue, can be used in the present methods. Solid supports for embedding fixed tissue can be removed with organic solvents to enable subsequent rehydration of preserved tissue.

In some cases, the assay includes a step of cell or tissue culture. For example, cells from a biopsy can be disaggregated using enzymes (such as collagenase and hyaluronidase) and or physical disruption (e.g., repeated passage through a 25-gauge needle) to dissociate the cells, collected by centrifugation, and resuspended in desired buffer or culture medium for culture, immediate analysis, or further processing.

Subject/Patient Population

In some embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human, who has or is diagnosed with a liquid cancer. In other embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human, predisposed or susceptible to a liquid cancer. In some embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human, at risk of developing a liquid cancer.

In some embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human, who has or is diagnosed with a liquid cancer, determined to lack a p53 deactivating mutation and/or expressing wild type p53. In other embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human, predisposed or susceptible to a liquid cancer, determined to lack a p53 deactivating mutation and/or expressing wild type p53. In some embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human, at risk of developing a liquid cancer, determined to lack a p53 deactivating mutation and/or expressing wild type p53. A p53 deactivating mutation, as used herein is any mutation that leads to loss of (or a decrease in) the in vitro apoptotic activity of p53. Non limiting examples of p53 deactivating mutations are included in Table 1. Accordingly, in some embodiments, a subject with a liquid cancer in accordance with the composition as provided herein is a human who has or is diagnosed with a liquid cancer that is determined to lack a p53 deactivation mutation, such as those shown in Table 1.

In some embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human, who has or is diagnosed with a liquid cancer, determined to lack a dominant p53 deactivating mutation. Dominant p53 deactivating mutation or dominant negative mutation, as used herein, is a mutation wherein the mutated p53 inhibits or disrupt the activity of the wild-type p53 gene.

TABLE 1 Examples of p53 deactivating mutations Mutation at position Amino acid change 62 E62_W91del 122 V122X 135 C135S 143 V143A 144 Q144P 146 W146X 157 V157F 158 R158H 163 Y163N 168 H168Y 173 V173L 175 R175H 175 R175P 175 R175Q 175 R175S 219 P219H 234 Y234C 234 Y234H 237 M237I 240 S240R 245 G245C 245 G245S 246 M246I 248 R248Q 248 R248W 249 R249S 272 V272M 273 R273H 274 V274F 279 G279E 280 R280K 281 D281H 282 R282W 306 R306P 308 P300_L308del 327 P300_Y327del 332 D324_I332del 337 R337C 344 L344P

Table 1 refers to the sequence of the wild-type human TP53 tumor protein p53 shown in FIG. 1. Amino acid changes are reported as: the amino acid being substituted followed by the position of the amino acid being substituted in the wild type p53 sequence, followed by the amino acid used for substitution. For example L344P, indicates that the leucine residue (L) at the 344 position in the wild type sequence is replaced by a proline residue (P).

In some embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a refractory patient. In a certain embodiment, a refractory patient is a patient refractory to a standard therapy (e.g., surgery, radiation, anti-androgen therapy and/or drug therapy such as chemotherapy). In certain embodiments, a patient with the liquid cancer is refractory to a therapy when the liquid cancer has not significantly been eradicated and/or the one or more symptoms have not been significantly alleviated. The determination of whether a patient is refractory can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of a treatment of liquid cancer. In various embodiments, a patient with liquid cancer is refractory when the number of CTCs or MNBCs associated with the liquid cancer have not decreased or have increased. In various embodiments, a patient with liquid cancer is refractory when one or more liquid cancer cells metastasize and/or spread to another organ.

In some embodiments, a subject treated for liquid cancer accordance with the methods provided herein is a human that has proven refractory to therapies other than treatment with the peptidomimetic macrocycles of the disclosure, but is no longer on these therapies. In certain embodiments, a subject treated for liquid cancer in accordance with the methods provided herein is a human already receiving one or more conventional anti-cancer therapies, such as surgery, drug therapy such as chemotherapy, anti-androgen therapy or radiation. Among these patients are refractory patients, patients who are too young for conventional therapies, and patients with recurring liquid cancer cells despite treatment with existing therapies.

In some embodiments, the subject is a human who has had at least one unsuccessful prior treatment and/or therapy of the liquid cancer.

Methods of Detecting Wild Type p53 and/or p53 Mutations

The liquid cancer cell samples from a subject can be assayed in order to determine the lack of a p53 deactivating mutation and/or expression of wild type p53.

In order to detect the p53 wild-type gene and/or lack of p53 deactivation mutation in a tissue, it can be helpful to isolate the tissue free from surrounding normal tissues. For example, the tissue can be isolated from paraffin or cryostat sections. Cancer cells can also be separated from normal cells by flow cytometry. If the liquid cancer cells tissue is highly contaminated with normal cells, detection of mutations can be more difficult.

Detection of point mutations can be accomplished by molecular cloning of the p53 allele (or alleles) present in the liquid cancer cell tissue and sequencing that allele(s). Alternatively, the polymerase chain reaction can be used to amplify p53 gene sequences directly from a genomic DNA preparation from the liquid cancer cell tissue. The DNA sequence of the amplified sequences can then be determined. See e.g., Saiki et al., Science, Vol. 239, p. 487, 1988; U.S. Pat. Nos. 4,683,202; and 4,683,195.

Specific deletions of p53 genes can also be detected. For example, restriction fragment length polymorphism (RFLP) probes for the p53 gene or surrounding marker genes can be used to score loss of a p53 allele.

Loss of wild-type p53 genes can also be detected on the basis of the loss of a wild-type expression product of the p53 gene. Such expression products include both the mRNA as well as the p53 protein product itself. Point mutations can be detected by sequencing the mRNA directly or via molecular cloning of cDNA made from the mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques. The cDNA can also be sequenced via the polymerase chain reaction (PCR).

Alternatively, mismatch detection can be used to detect point mutations in the p53 gene or its mRNA product. The method can involve the use of a labeled riboprobe which is complementary to the human wild-type p53 gene. The riboprobe and either mRNA or DNA isolated from the liquid cancer cell tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full-length duplex RNA for the riboprobe and the p53 mRNA or DNA. The riboprobe need not be the full length of the p53 mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the p53 mRNA or gene it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton et al., Proc. Natl. Acad. Sci. USA, vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad. Sci. USA, vol. 72, p. 989, 1975. Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, Human Genetics, vol. 42, p. 726, 1988. With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization.

DNA sequences of the p53 gene from the liquid cancer cell tissue which have been amplified by use of polymerase chain reaction can also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the p53 gene sequence harboring a known mutation. For example, one oligomer can be about 30 nucleotides in length, corresponding to a portion of the p53 gene sequence. At the position coding for the 175th codon of p53 gene the oligomer encodes an alanine, rather than the wild-type codon valine. By use of a battery of such allele-specific probes, the PCR amplification products can be screened to identify the presence of a previously identified mutation in the p53 gene. Hybridization of allele-specific probes with amplified p53 sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe indicates the presence of the same mutation in the liquid cancer cell tissue as in the allele-specific probe.

The identification of p53 gene structural changes in liquid cancer cells can be facilitated through the application of a diverse series of high resolution, high throughput microarray platforms. Essentially there are two types of array; those that carry PCR products from cloned nucleic acids (e.g. cDNA, BACs, cosmids) and those that use oligonucleotides. The methods can provide a way to survey genome wide DNA copy number abnormalities and expression levels to allow correlations between losses, gains and amplifications in liquid cancer cells with genes that are over- and under-expressed in the same samples. The gene expression arrays that provide estimates of mRNA levels in liquid cancer cells have given rise to exon-specific arrays that can identify both gene expression levels, alternative splicing events and mRNA processing alterations. Oligonucleotide arrays are also being used to interrogate single nucleotide polymorphisms (SNPs) throughout the genome for linkage and association studies and these have been adapted to quantify copy number abnormalities and loss of heterozygosity events. DNA sequencing arrays can allow resequencing of chromosome regions and whole genomes.

SNP-based arrays or other gene arrays or chips are also contemplated to determine the presence of wild-type p53 allele and the structure of mutations. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome. For example, there are an estimated 5-10 million SNPs in the human genome. As SNPs are highly conserved throughout evolution and within a population, the map of SNPs serves as an excellent genotypic marker for research. SNP array is a useful tool to study the whole genome.

In addition, SNP array can be used for studying the Loss Of Heterozygosity (LOH). LOH is a form of allelic imbalance that can result from the complete loss of an allele or from an increase in copy number of one allele relative to the other. While other chip-based methods (e.g., comparative genomic hybridization can detect only genomic gains or deletions), SNP array has the additional advantage of detecting copy number neutral LOH due to uniparental disomy (UPD). In UPD, one allele or whole chromosome from one parent are missing leading to reduplication of the other parental allele (uni-parental=from one parent, disomy=duplicated). In a disease setting this occurrence can be pathologic when the wild-type allele (e.g., from the mother) is missing and instead two copies of the heterozygous allele (e.g., from the father) are present. This usage of SNP array has a huge potential in cancer diagnostics as LOH is a prominent characteristic of most human cancers. SNP array technology have shown that not only liquid cancers (e.g. gastric cancer, liver cancer etc) but also hematologic malignancies (ALL, MDS, CML etc) have a high rate of LOH due to genomic deletions or UPD and genomic gains. In the present disclosure, using high density SNP array to detect LOH allows identification of pattern of allelic imbalance to determine the presence of wild-type p53 allele (Lips et ah, 2005; Lai et al, 2007).

Examples for current p53 gene sequence and single nucleotide polymorphism arrays include p53 Gene Chip (Affymetrix, Santa Clara, Calif.), Roche p53 Ampli-Chip (Roche Molecular Systems, Pleasanton, Calif.), GeneChip Mapping arrays (Affymetrix, Santa Clara, Calif.), SNP Array 6.0 (Affymetrix, Santa Clara, Calif.), BeadArrays (Illumina, San Diego, Calif.), etc.

Mutations of wild-type p53 genes can also be detected on the basis of the mutation of a wild-type expression product of the p53 gene. Such expression products include both the mRNA as well as the p53 protein product itself. Point mutations can be detected by sequencing the mRNA directly or via molecular cloning of cDNA made from the mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques. The cDNA can also be sequenced via the polymerase chain reaction (PCR). A panel of monoclonal antibodies could be used in which each of the epitopes involved in p53 functions are represented by a monoclonal antibody. Loss or perturbation of binding of a monoclonal antibody in the panel can indicate mutational alteration of the p53 protein and thus of the p53 gene itself. Mutant p53 genes or gene products can also be detected in body samples, such as, serum, stool, or other body fluids, such as urine and sputum. The same techniques discussed above for detection of mutant p53 genes or gene products in tissues can be applied to other body samples.

Loss of wild-type p53 genes can also be detected by screening for loss of wild-type p53 protein function. Although all of the functions which the p53 protein undoubtedly possesses have yet to be elucidated, at least two specific functions are known. Protein p53 binds to the SV40 large T antigen as well as to the adenovirus E1B antigen. Loss of the ability of the p53 protein to bind to either or both of these antigens indicates a mutational alteration in the protein which reflects a mutational alteration of the gene itself. Alternatively, a panel of monoclonal antibodies could be used in which each of the epitopes involved in p53 functions are represented by a monoclonal antibody. Loss or perturbation of binding of a monoclonal antibody in the panel would indicate mutational alteration of the p53 protein and thus of the p53 gene itself. Any means for detecting an altered p53 protein can be used to detect loss of wild-type p53 genes.

Mutant p53 genes or gene products can also be detected in body samples, such as, serum, stool, or other body fluids, such as urine and sputum. The same techniques discussed above for detection of mutant p53 genes or gene products in tissues can be applied to other body samples.

Determination of the lack of p53 deactivating mutation and/or expression of wild type p53 in the subject with liquid cancer can be performedd any time before, during or after the administration of the peptidomimetic macrocycles. In some embodiments, the determination of the lack of a p53 deactivating mutation and/or expression of wild type p53 is performed before the first administration of the peptidomimetic macrocycle to the subject, for example about 5 years- about 1 month, about 4 years- about lmonth, about 3 years-1 month, about 2 years- about 1 month, about 1 years- about 1 month, about 5 years- about 1 week, about 4 years- about 1 week, about 3 years- about 1 month, about 2 years- about 1 week, about 1 year- about 1 week, about 5 years- about 1 day, about 4 years- about 1 day, about 3 years- about 1 day, about 2 years- about Iday, about 1 year- about 1 day, about 15 months- about 1 month, about 15 months- about 1 week, about 15 months- about 1 day, about 12 months- about 1 month, about 12 months- about 1 week, about 12 months- about 1 day, about 6 months-1 about month, about 6 months- about 1 week, about 6 months- about 1 day, about 3 months-1 about month, about 3 months- about 1 week, or about 3 months- about 1 day prior to the first administration of the peptidomimetic macrocycle to the subject. In some examples, the confirmation of the lack of the p53 deactivating mutation and/or expression of wild type p53 is performed up to 6 years, 5 years, 4 years, 3 years, 24 months, 23 months, 22 months, 21 months, 20 months, 19 months, 18 months, 17 months, 16 months, 15 months, 14 months, 13 months, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 months, 4 weeks (28 days), 3 weeks (21 days), 2 weeks (14 days), 1 week (7 days), 6 days, 5 days, 4 days, 3 days, 2 days or 1 day before the first administration of the peptidomimetic macrocycle to the subject. In some examples the confirmation of the lack of the p53 deactivating mutation is performed within 1 month of the first administration of the peptidomimetic macrocycle to the subject. In some examples the confirmation of the lack of the p53 deactivating mutation is performed within 21 days of the first administration of the peptidomimetic macrocycle to the subject.

Liquid Cancers

Liquid cancers that can be treated by the instant methods include, but are not limited to, liquid lymphomas, lekemias, and myelomas. Examples of liquid lymphomas and leukemias that can be treated in accordance with the methods described include, but are not limited to, chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma, classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte depleted or not depleted), and nodular lymphocyte-predominant Hodgkin lymphoma.

Examples of liquid cancers that can be treated by the methods of the disclosure include cancers involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Examples of disorders include: acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), multiple mylenoma, hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease. For example, liquid cancers include, but are not limited to, acute lymphocytic leukemia (ALL); T-cell acute lymphocytic leukemia (T-ALL); anaplastic large cell lymphoma (ALCL); chronic myelogenous leukemia (CML); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); B-cell chronic lymphocytic leukemia (B-CLL); diffuse large B-cell lymphomas (DLBCL); hyper eosinophilia/chronic eosinophilia; and Burkitt's lymphoma.

In some embodiments, the liquid cancer treated by the methods of the disclosure is an acute lymphoblastic leukemia; acute myeloid leukemia; AIDS-related cancers; AIDS-related lymphoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; cutaneous T-cell lymphoma; Hodgkin lymphoma; multiple myeloma; multiple myeloma/plasma cell neoplasm; Non-Hodgkin lymphoma; primary central nervous system (CNS) lymphoma; or T-cell lymphoma; In various embodiments, the liquid cancer can be B-Cell Chronic Lymphocytic Leukemia, B-Cell Lymphoma-DLBCL, B-Cell Lymphoma-DLBCL-germinal center-like, B-Cell Lymphoma-DLBCL-activated B-cell-like, or Burkitt's lymphoma.

In some embodiments liquid cancers treated by the methods disclosed herein exclude cancers that are known to be associated with HPV (Human papillomavirus).

The effectiveness and/or response of cancer treatment by the methods disclosed herein can be determined by any suitable method. The response can be a complete response, and which can be an objective response, a clinical response, or a pathological response to treatment. For example, the response can be determined based upon the techniques for evaluating response to treatment of liquid cancers as described in or by Revised International Working Group Response Criteria for liquid lymphoma patients (IWG 2014), which is hereby incorporated by reference in its entirety. The response can be a duration of survival (or probability of such duration) or progression-free interval. The timing or duration of such events can be determined from about the time of diagnosis, or from about the time treatment is initiated or from about the time treatment is finished (like the final administration of the peptidomimetic macrocycle). Alternatively, the response can be based upon a reduction in the number of liquid cancer cells, the number of liquid cancer cells per unit volume, or liquid cancer cell metabolism, or based upon overall liquid cancer cell burden, or based upon levels of serum markers especially where elevated in the disease state.

The response in individual patients can be characterized as a complete response, a partial response, stable disease, and progressive disease. In some embodiments, the response is complete response (CR). Complete response can be defined as disappearance of all circulating tumor cells (CTC) or a mononuclear blood cells (MNBC) i.e. any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. In some examples (e.g., AML), complete response can be defined as the following: bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; absolute neutrophil count >1.0×109/L (1000/μL); platelet count >100×109/L (100 000/μL); and independence of red cell transfusions. In certain embodiments, the response is a CR with Incomplete Recovery (CRi). CR with Incomplete Recovery, in some examples (e.g., AML), can be defined to include all CR criteria except for residual neutropenia (<1.0×109/L [1000/μL]) or thrombocytopenia (<100×109/L [100 000/μL]). In certain embodiments, the response is a morphologic leukemia free state. Morphologic leukemia free state, in some examples (e.g., AML), can be defined to include bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; and no hematologic recovery required. In certain embodiments, the response is a partial response (PR). Partial response can be defined to mean at least 30% decrease in the sum of diameters of circulating tumor cells (CTC) or a mononuclear blood cells (MNBC), taking as reference the baseline sum diameters. In some examples (e.g., AML), partial response can be defined to include all hematologic criteria of CR; decrease of bone marrow blast percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50%. In certain embodiments, the response is a morphologic leukemia free state. Morphologic leukemia free state, in some examples (e.g., AML), can be defined to include bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; and no hematologic recovery required. In certain embodiments, the response is a relapse. Relapse, in some examples (e.g., AML), can be defined to include bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; and no hematologic recovery required. In some embodiments, the response is progressive disease (PD). Progressive disease can be defined as at least a 20% increase in the number of circulating tumor cells (CTC) or a mononuclear blood cells (MNBC), taking as reference the smallest number on study (this includes the baseline number if that is the smallest) and an absolute increase of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, or at least 100 or more circulating tumor cells (CTC) or a mononuclear blood cells (MNBC). The appearance of one or more new lesions can also be considered as progression. In some embodiments, the disease can be stable disease (SD). Stable disease can be characterized by neither sufficient decrease in liquid cancer cell number to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest number of CTCs and/or MNBCs while on study. In certain embodiments, the response is a pathological complete response. A pathological complete response, e.g., as determined by a pathologist following examination of tissue removed at the time of surgery or biopsy, generally refers to an absence of histological evidence of invasive and/or non-invasive liquid cancer cells in the surgical specimen.

Combination Treatment

Also provided herein are combination therapies for the treatment of a liquid cancer which involve the administration of the peptidomimetic macrocycles disclosed herein in combination with one or more additional therapies to a subject with liquid cancer determined to lack a p53 deactivating mutation and/or express wild type p53. In a specific embodiment, presented herein are combination therapies for the treatment of liquid cancer which involve the administration of an effective amount of the peptidomimetic macrocycles in combination with an effective amount of another therapy to a subject with a liquid cancer determined to lack a p53 deactivating mutation and/or with a liquid cancer expressing wild type p53.

As used herein, the term “in combination,” refers, in the context of the administration of the peptidomimetic macrocycles, to the administration of the peptidomimetic macrocycles prior to, concurrently with, or subsequent to the administration of one or more additional therapies (e.g., agents, surgery, or radiation) for use in treating liquid cancer. The use of the term “in combination” does not restrict the order in which the peptidomimetic macrocycles and one or more additional therapies are administered to a subject. In specific embodiments, the interval of time between the administration of the peptidomimetic macrocycles and the administration of one or more additional therapies can be about 1- about 5 minutes, about 1- about 30 minutes, about 30 minutes to about 60 minutes, about 1 hour, about 1- about 2 hours, about 2- about 6 hours, about 2- about 12 hours, about 12- about 24 hours, about 1- about 2 days, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 15 weeks, about 20 weeks, about 26 weeks, about 52 weeks, about 11- about 15 weeks, about 15- about 20 weeks, about 20- about 30 weeks, about 30- about 40 weeks, about 40- about 50 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 2 years, or any period of time in between. In certain embodiments, the peptidomimetic macrocycles and one or more additional therapies are administered less than 1 day, less than 1 week, less than 2 weeks, less than 3 weeks, less than 4 weeks, less than one month, less than 2 months, less than 3 months, less than 6 months, less than 1 year, less than 2 years, or less than 5 years apart.

In some embodiments, the combination therapies provided herein involve administering of the peptidomimetic macrocycles 1-2 times a week, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks or once every 8 weeks and administering one or more additional therapies once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every month, once every 2 months (e.g., approximately 8 weeks), once every 3 months (e.g., approximately 12 weeks), or once every 4 months (e.g., approximately 16 weeks). In certain embodiments, the peptidomimetic macrocycles and one or more additional therapies are cyclically administered to a subject. Cycling therapy involves the administration of the peptidomimetic macrocycles compounds for a period of time, followed by the administration of one or more additional therapies for a period of time, and repeating this sequential administration. In certain embodiments, cycling therapy can also include a period of rest where the peptidomimetic macrocycles or the additional therapy is not administered for a period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years). In an embodiment, the number of cycles administered is from 1 to 12 cycles, from 2 to 10 cycles, or from 2 to 8 cycles.

In some embodiments, the methods for treating liquid cancer provided herein comprise administering the peptidomimetic macrocycles as a single agent for a period of time prior to administering the peptidomimetic macrocycles in combination with an additional therapy. In certain embodiments, the methods for treating cancer provided herein comprise administering an additional therapy alone for a period of time prior to administering the peptidomimetic macrocycles in combination with the additional therapy.

In some embodiments, the administration of the peptidomimetic macrocycles and one or more additional therapies in accordance with the methods presented herein have an additive effect relative the administration of the peptidomimetic macrocycles or said one or more additional therapies alone. In some embodiments, the administration of the peptidomimetic macrocycles and one or more additional therapies in accordance with the methods presented herein have a synergistic effect relative to the administration of the peptidomimetic macrocycles or said one or more additional therapies alone.

As used herein, the term “synergistic,” refers to the effect of the administration of the peptidomimetic macrocycles in combination with one or more additional therapies (e.g., agents), which combination is more effective than the additive effects of any two or more single therapies (e.g., agents). In a specific embodiment, a synergistic effect of a combination therapy permits the use of lower dosages (e.g., sub-optimal doses) of the peptidomimetic macrocycles or an additional therapy and/or less frequent administration of the peptidomimetic macrocycles or an additional therapy to a subject. In certain embodiments, the ability to utilize lower dosages of the peptidomimetic macrocycles or of an additional therapy and/or to administer the peptidomimetic macrocycles or said additional therapy less frequently reduces the toxicity associated with the administration of the peptidomimetic macrocycles or of said additional therapy, respectively, to a subject without reducing the efficacy of the peptidomimetic macrocycles or of said additional therapy, respectively, in the treatment of liquid cancer. In some embodiments, a synergistic effect results in improved efficacy of the peptidomimetic macrocycles and each of said additional therapies in treating cancer. In some embodiments, a synergistic effect of a combination of the peptidomimetic macrocycles and one or more additional therapies avoids or reduces adverse or unwanted side effects associated with the use of any single therapy.

The combination of the peptidomimetic macrocycles and one or more additional therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the peptidomimetic macrocycles and one or more additional therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The peptidomimetic macrocycles and one or more additional therapies can be administered sequentially to a subject in separate pharmaceutical compositions. The peptidomimetic macrocycles compounds and one or more additional therapies can also be administered to a subject by the same or different routes of administration.

The combination therapies provided herein involve administering to a subject to in need thereof the peptidomimetic macrocycles in combination with conventional, or known, therapies for treating cancer. Other therapies for cancer or a condition associated therewith are aimed at controlling or relieving one or more symptoms. Accordingly, in some embodiments, the combination therapies provided herein involve administering to a subject to in need thereof a pain reliever, or other therapies aimed at alleviating or controlling one or more symptoms associated with or a condition associated therewith.

Non-limiting specific examples of anti-cancer agents that can be used in combination with the peptidomimetic macrocycles include: a hormonal agent (e.g., aromatase inhibitor, selective estrogen receptor modulator (SERM), and estrogen receptor antagonist), chemotherapeutic agent (e.g., microtubule disassembly blocker, antimetabolite, topoisomerase inhibitor, and DNA crosslinker or damaging agent), anti-antigenic agent (e.g., VEGF antagonist, receptor antagonist, integrin antagonist, vascular targeting agent (VTA)/vascular disrupting agent (VDA)), radiation therapy, and conventional surgery.

Non-limiting examples of hormonal agents that can be used in combination with the peptidomimetic macrocycles include aromatase inhibitors, SERMs, and estrogen receptor antagonists. Hormonal agents that are aromatase inhibitors can be steroidal or no steroidal. Non-limiting examples of no steroidal hormonal agents include letrozole, anastrozole, aminoglutethimide, fadrozole, and vorozole. Non-limiting examples of steroidal hormonal agents include aromasin (exemestane), formestane, and testolactone. Non-limiting examples of hormonal agents that are SERMs include tamoxifen (branded/marketed as Nolvadex®), afimoxifene, arzoxifene, bazedoxifene, clomifene, femarelle, lasofoxifene, ormeloxifene, raloxifene, and toremifene. Non-limiting examples of hormonal agents that are estrogen receptor antagonists include fulvestrant. Other hormonal agents include but are not limited to abiraterone and lonaprisan.

Non-limiting examples of chemotherapeutic agents that can be used in combination with of peptidomimetic macrocycles include microtubule disassembly blocker, antimetabolite, topoisomerase inhibitor, and DNA crosslinker or damaging agent. Chemotherapeutic agents that are microtubule disassembly blockers include, but are not limited to, taxanes (e.g., paclitaxel (branded/marketed as TAXOL®), docetaxel, abraxane, larotaxel, ortataxel, and tesetaxel); epothilones (e.g., ixabepilone); and vinca alkaloids (e.g., vinorelbine, vinblastine, vindesine, and vincristine (branded/marketed as ONCOVIN®)).

Chemotherapeutic agents that are antimetabolites include, but are not limited to, folate anitmetabolites (e.g., methotrexate, aminopterin, pemetrexed, raltitrexed); purine antimetabolites (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine); pyrimidine antimetabolites (e.g., 5-fluorouracil, capcitabine, gemcitabine (GEMZAR®), cytarabine, decitabine, floxuridine, tegafur); and deoxyribonucleotide antimetabolites (e.g., hydroxyurea).

Chemotherapeutic agents that are topoisomerase inhibitors include, but are not limited to, class I (camptotheca) topoisomerase inhibitors (e.g., topotecan (branded/marketed as HYCAMTIN®) irinotecan, rubitecan, and belotecan); class II (podophyllum) topoisomerase inhibitors (e.g., etoposide or VP-16, and teniposide); anthracyclines (e.g., doxorubicin, epirubicin, Doxil, aclarubicin, amrubicin, daunorubicin, idarubicin, pirarubicin, valrubicin, and zorubicin); and anthracenediones (e.g., mitoxantrone, and pixantrone).

Chemotherapeutic agents that are DNA crosslinkers (or DNA damaging agents) include, but are not limited to, alkylating agents (e.g., cyclophosphamide, mechlorethamine, ifosfamide (branded/marketed as IFEX®), trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, carmustine (branded/marketed as BiCNU®), lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, busulfan, mannosulfan, treosulfan, carboquone, N,N′N′-triethylenethiophosphoramide, triaziquone, triethylenemelamine); alkylating-like agents (e.g., carboplatin (branded/marketed as PARAPLATIN®), cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, satraplatin, picoplatin); nonclassical DNA crosslinkers (e.g., procarbazine, dacarbazine, temozolomide (branded/marketed as TEMODAR®), altretamine, mitobronitol); and intercalating agents (e.g., actinomycin, bleomycin, mitomycin, and plicamycin).

Non-limiting examples of other therapies that can be administered to a subject in combination with the peptidomimetic macrocycles include: (1) a statin such as lovostatin (e.g., branded/marketed as MEVACOR®); (2) an mTOR inhibitor such as sirolimus which is also known as Rapamycin (e.g., branded/marketed as RAPAMUNE®), temsirolimus (e.g., branded/marketed as TORISEL®), evorolimus (e.g., branded/marketed as AFINITOR®), and deforolimus; (3) a farnesyltransferase inhibitor agent such as tipifarnib; (4) an antifibrotic agent such as pirfenidone; (5) a pegylated interferon such as PEG-interferon alfa-2b; (6) a CNS stimulant such as methylphenidate (branded/marketed as RITALIN®); (7) a HER-2 antagonist such as anti-HER-2 antibody (e.g., trastuzumab) and kinase inhibitor (e.g., lapatinib); (8) an IGF-1 antagonist such as an anti-IGF-1 antibody (e.g., AVE1642 and IMC-A11) or an IGF-1 kinase inhibitor; (9) EGFR/HER-1 antagonist such as an anti-EGFR antibody (e.g., cetuximab, panitumamab) or EGFR kinase inhibitor (e.g., erlotinib; gefitinib); (10) SRC antagonist such as bosutinib; (11) cyclin dependent kinase (CDK) inhibitor such as seliciclib; (12) Janus kinase 2 inhibitor such as lestaurtinib; (13) proteasome inhibitor such as bortezomib; (14) phosphodiesterase inhibitor such as anagrelide; (15) inosine monophosphate dehydrogenase inhibitor such as tiazofurine; (16) lipoxygenase inhibitor such as masoprocol; (17) endothelin antagonist; (18) retinoid receptor antagonist such as tretinoin or alitretinoin; (19) immune modulator such as lenalidomide, pomalidomide, or thalidomide; (20) kinase (e.g., tyrosine kinase) inhibitor such as imatinib, dasatinib, erlotinib, nilotinib, gefitinib, sorafenib, sunitinib, lapatinib, or TG100801; (21) non-steroidal anti-inflammatory agent such as celecoxib (branded/marketed as CELEBREX®); (22) human granulocyte colony-stimulating factor (G-CSF) such as filgrastim (branded/marketed as NEUPOGEN®); (23) folinic acid or leucovorin calcium; (24) integrin antagonist such as an integrin a53l1-antagonist (e.g., JSM6427); (25) nuclear factor kappa beta (NF-Kx) antagonist such as OT-551, which is also an anti-oxidant. (26) hedgehog inhibitor such as CUR61414, cyclopamine, GDC-0449, and anti-hedgehog antibody; (27) histone deacetylase (HDAC) inhibitor such as SAHA (also known as vorinostat (branded/marketed as ZOLINZA)), PCI-24781, SB939, CHR-3996, CRA-024781, ITF2357, JNJ-26481585, or PCI-24781; (28) retinoid such as isotretinoin (e.g., branded/marketed as ACCUTANE®); (29) hepatocyte growth factor/scatter factor (HGF/SF) antagonist such as HGF/SF monoclonal antibody (e.g., AMG 102); (30) synthetic chemical such as antineoplaston; (31) anti-diabetic such as rosaiglitazone (e.g., branded/marketed as AVANDIA®); (32) antimalarial and amebicidal drug such as chloroquine (e.g., branded/marketed as ARALEN®); (33) synthetic bradykinin such as RMP-7; (34) platelet-derived growth factor receptor inhibitor such as SU-101; (35) receptor tyrosine kinase inhibitorsof Flk-1/KDR/VEGFR2, FGFR1 and PDGFR beta such as SU5416 and SU6668; (36) anti-inflammatory agent such as sulfasalazine (e.g., branded/marketed as AZULFIDINE®); and (37) TGF-beta antisense therapy.

In some embodiments the peptidomimetic macrocycles disclosed herein can inhibit one or more transporter enzymes (e.g., OATP1B1, OATP1B3, BSEP) at concentrations that can be clinically relevant. Therefore the peptidomimetic macrocycles disclosed herein can interact with medications that are predominantly cleared by hepatobiliary transporters. In particular, methotrexate and statins (e.g., atorvastatin, fluvastatin lovastatin, pitavastatin pravastatin, rosuvastatin and simvastatin) can not be dosed within 48 h, 36 h, 24 h, or 12 h ((for example within 24 h) of the administration of the peptidomimetic macrocycles disclosed herein. Examples of medications that can be affected by co-administration with peptidomimetic macrocycles disclosed herein are listed below. In various embodiments one or more of the medications selected from Table 1 is not dosed within 48 h, 36 h, 24 h, or 12 h (for example within 24 h) of the administration of the peptidomimetic macrocycles disclosed herein.

TABLE 2 Exemple medications that can be affected by co-administration with peptidomimetic macrocycles disclosed herein. Medication Therapeutic Area Irinotecan Oncology Bosentan Pulmonary artery hypertension Caspofungin Antifungal Methotrexate Oncology & rheumatology Repaglinide Diabetes mellitus Atorvastatin Hypercholesterolemia Cerivastatin Hypercholesterolemia Fluvastatin Hypercholesterolemia Lovastatin Hypercholesterolemia Pitavastatin Hypercholesterolemia Pravastatin Hypercholesterolemia Rosuvastatin Hypercholesterolemia Simvastatin Hypercholesterolemia

Biological Samples

As used in the present application, “biological sample” means any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus, and the like. Also included within the meaning of the term “biological sample” is an organ or tissue extract and culture fluid in which any cells or tissue preparation from a subject has been incubated. The biological samples can be any samples from which genetic material can be obtained. Biological samples can also include solid or liquid cancer cell samples or specimens. The cancer cell sample can be a cancer cell tissue sample. In some embodiments, the cancer cell tissue sample can obtained from surgically excised tissue. Exemplary sources of biological samples include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In some cases, the biological samples comprise fine needle aspiration samples. In some embodiments, the biological samples comprise tissue samples, including, for example, excisional biopsy, incisional biopsy, or other biopsy. The biological samples can comprise a mixture of two or more sources; for example, fine needle aspirates and tissue samples. Tissue samples and cellular samples can also be obtained without invasive surgery, for example by punctuating the chest wall or the abdominal wall or from masses of breast, thyroid or other sites with a fine needle and withdrawing cellular material (fine needle aspiration biopsy). In some embodiments, a biological sample is a bone marrow aspirate sample. A biological sample can be obtained by biopsy methods provided herein, swabbing, scraping, phlebotomy, or any other suitable method.

Methods of Detecting Wild Type p53 and/or p53 Mutations

In some embodiments, a subject lacking p53-deactivating mutations is a candidate for cancer treatment with a compound of the invention. Cancer cells from patient groups should be assayed in order to determine p53-deactivating mutations and/or expression of wild type p53 prior to treatment with a compound of the invention.

The activity of the p53 pathway can be determined by the mutational status of genes involved in the p53 pathways, including, for example, AKT1, AKT2, AKT3, ALK, BRAF, CDK4, CDKN2A, DDR2, EGFR, ERBB2 (HER2), FGFR1, FGFR3, GNA11, GNQ, GNAS, KDR, KIT, KRAS, MAP2K1 (MEK1), MET, HRAS, NOTCH1, NRAS, NTRK2, PIK3CA, NF1, PTEN, RAC1, RB1, NTRK3, STK11, PIK3R1, TSC1, TSC2, RET, TP53, and VHL. Genes that modulate the activity of p53 can also be assessed, including, for example, kinases: ABL1, JAK1, JAAK2, JAK3; receptor tyrosine kinases: FLT3 and KIT; receptors: CSF3R, IL7R, MPL, and NOTCH1; transcription factors: BCOR, CEBPA, CREBBP, ETV6, GATA1, GATA2. MLL, KZF1, PAX5, RUNX1, STAT3, WT1, and TP53; epigenetic factors: ASXL1, DNMT3A, EZH2, KDM6A (UTX), SUZ12, TET2, PTPN11, SF3B1, SRSF2, U2AF35, ZRSR2; RAS proteins: HRAS, KRAS, and NRAS; adaptors CBL and CBL-B; FBXW7, IDH1, IDH2, and NPM1.

Cancer cell samples can be obtained, for example, from solid or liquid tumors via primary or metastatic tumor resection (e.g. pneumonectomy, lobetomy, wedge resection, and craniotomy) primary or metastatic disease biopsy (e.g. transbronchial or needle core), pleural or ascites fluid (e.g. FFPE cell pellet), bone marrow aspirate, bone marrow clot, and bone marrow biopsy, or macro-dissection of tumor rich areas (solid tumors).

To detect the p53 wild type gene and/or lack of p53 deactivation mutation in a tissue, cancerous tissue can be isolated from surrounding normal tissues. For example, the tissue can be isolated from paraffin or cryostat sections. Cancer cells can also be separated from normal cells by flow cytometry. If the cancer cells tissue is highly contaminated with normal cells, detection of mutations can be more difficult.

Various methods and assays for analyzing wild type p53 and/or p53 mutations are suitable for use in the invention. Non-limiting examples of assays include polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), microarray, Southern Blot, Northern Blot, Western Blot, Eastern Blot, H&E staining, microscopic assessment of tumors, next-generation DNA sequencing (NGS) (e.g. extraction, purification, quantification, and amplification of DNA, library preparation) immunohistochemistry, and fluorescent in situ hybridization (FISH).

A microarray allows a researcher to investigate multiple DNA sequences attached to a surface, for example, a DNA chip made of glass or silicon, or a polymeric bead or resin. The DNA sequences are hybridized with fluorescent or luminescent probes. The microarray can indicate the presence of oligonucleotide sequences in a sample based on hybridization of sample sequences to the probes, followed by washing and subsequent detection of the probes. Quantification of the fluorescent or luminescent signal indicates the presence of known oligonucleotide sequences in the sample.

PCR allows amplification of DNA oligomers rapidly, and can be used to identify an oligonucleotide sequence in a sample. PCR experiments involve contacting an oligonucleotide sample with a PCR mixture containing primers complementary to a target sequence, one or more DNA polymerase enzymes, deoxnucleotide triphosphate (dNTP) building blocks, including dATP, dGTP, dTTP, and dCTP, and suitable buffers, salts, and additives. If a sample contains an oligonucleotide sequence complementary to a pair of primers, the experiment amplifies the sample sequence, which can be collected and identified.

In some embodiments, an assay comprises amplifying a biomolecule from the cancer sample. The biomolecule can be a nucleic acid molecule, such as DNA or RNA. In some embodiments, the assay comprises circularization of a nucleic acid molecule, followed by digestion of the circularized nucleic acid molecule.

In some embodiments, the assay comprises contacting an organism, or a biochemical sample collected from an organism, such as a nucleic acid sample, with a library of oligonucleotides, such as PCR primers. The library can contain any number of oligonucleotide molecules. The oligonucleotide molecules can bind individual DNA or RNA motifs, or any combination of motifs described herein. The motifs can be any distance apart, and the distance can be known or unknown. In some embodiments, two or more oligonucleotides in the same library bind motifs a known distance apart in a parent nucleic acid sequence. Binding of the primers to the parent sequence can take place based on the complementarity of the primers to the parent sequence. Binding can take place, for example, under annealing, or under stringent conditions.

In some embodiments, the results of an assay are used to design a new oligonucleotide sequence for future use. In some embodiments, the results of an assay are used to design a new oligonucleotide library for future use. In some embodiments, the results of an assay are used to revise, refine, or update an existing oligonucleotide library for future use. For example, an assay can reveal that a previously-undocumented nucleic acid sequence is associated with the presence of a target material. This information can be used to design or redesign nucleic acid molecules and libraries.

In some embodiments, one or more nucleic acid molecules in a library comprise a barcode tag. In some embodiments, one or more of the nucleic acid molecules in a library comprise type I or type II restriction sites suitable for circularization and cutting an amplified sample nucleic acid sequence. Such primers can be used to circularize a PCR product and cut the PCR product to provide a product nucleic acid sequence with a sequence that is organized differently from the nucleic acid sequence native to the sample organism.

After a PCR experiment, the presence of an amplified sequence can be verified. Non-limiting examples of methods for finding an amplified sequence include DNA sequencing, whole transcriptome shotgun sequencing (WTSS, or RNA-seq), mass spectrometry (MS), microarray, pyrosequencing, column purification analysis, polyacrylamide gel electrophoresis, and index tag sequencing of a PCR product generated from an index-tagged primer.

In some embodiments, more than one nucleic acid sequence in the sample organism is amplified. Non-limiting examples of methods of separating different nucleic acid sequences in a PCR product mixture include column purification, high performance liquid chromatography (HPLC), HPLC/MS, polyacrylamide gel electrophoresis, size exclusion chromatography.

The amplified nucleic acid molecules can be identified by sequencing. Nucleic acid sequencing can be done on automated instrumentation. Sequencing experiments can be done in parallel to analyze tens, hundreds, or thousands of sequences simultaneously. Non-limiting examples of sequencing techniques follow.

In pyrosequencing, DNA is amplified within a water droplet containing a single DNA template bound to a primer-coated bead in an oil solution. Nucleotides are added to a growing sequence, and the addition of each base is evidenced by visual light.

Ion semiconductor sequencing detects the addition of a nucleic acid residue as an electrical signal associated with a hydrogen ion liberated during synthesis. A reaction well containing a template is flooded with the four types of nucleotide building blocks, one at a time. The timing of the electrical signal identifies which building block was added, and identifies the corresponding residue in the template.

DNA nanoball uses rolling circle replication to amplify DNA into nanoballs. Unchained sequencing by ligation of the nanoballs reveals the DNA sequence.

In a reversible dyes approach, nucleic acid molecules are annealed to primers on a slide and amplified. Four types of fluorescent dye residues, each complementary to a native nucleobase, are added, the residue complementary to the next base in the nucleic acid sequence is added, and unincorporated dyes are rinsed from the slide. Four types of reversible terminator bases (RT-bases) are added, and non-incorporated nucleotides are washed away. Fluorescence indicates the addition of a dye residue, thus identifying the complementary base in the template sequence. The dye residue is chemically removed, and the cycle repeats.

Detection of point mutations can be accomplished by molecular cloning of the p53 allele(s) present in the cancer cell tissue and sequencing that allele(s). Alternatively, the polymerase chain reaction can be used to amplify p53 gene sequences directly from a genomic DNA preparation from the cancer cell tissue. The DNA sequence of the amplified sequences can then be determined. See e.g., Saiki et al., Science, Vol. 239, p. 487, 1988; U.S. Pat. Nos. 4,683,202; and 4,683,195. Specific deletions of p53 genes can also be detected. For example, restriction fragment length polymorphism (RFLP) probes for the p53 gene or surrounding marker genes can be used to score loss of a p53 allele.

Loss of wild type p53 genes can also be detected on the basis of the loss of a wild type expression product of the p53 gene. Such expression products include both the mRNA as well as the p53 protein product itself. Point mutations can be detected by sequencing the mRNA directly or via molecular cloning of cDNA made from the mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques. The cDNA can also be sequenced via the polymerase chain reaction (PCR).

Alternatively, mismatch detection can be used to detect point mutations in the p53 gene or the mRNA product. The method can involve the use of a labeled riboprobe that is complementary to the human wild type p53 gene. The riboprobe and either mRNA or DNA isolated from the cancer cell tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, the enzyme cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product is seen that is smaller than the full-length duplex RNA for the riboprobe and the p53 mRNA or DNA. The riboprobe need not be the full length of the p53 mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the p53 mRNA or gene it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton et al., Proc. Natl. Acad. Sci. USA, vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad. Sci. USA, vol. 72, p. 989, 1975. Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, Human Genetics, vol. 42, p. 726, 1988. With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization.

DNA sequences of the p53 gene from the cancer cell tissue which have been amplified by use of polymerase chain reaction can also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the p53 gene sequence harboring a known mutation. For example, one oligomer can be about 30 nucleotides in length, corresponding to a portion of the p53 gene sequence. At the position coding for the 175th codon of p53 gene the oligomer encodes an alanine, rather than the wild type codon valine. By use of a battery of such allele-specific probes, the PCR amplification products can be screened to identify the presence of a previously identified mutation in the p53 gene. Hybridization of allele-specific probes with amplified p53 sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe indicates the presence of the same mutation in the cancer cell tissue as in the allele-specific probe.

The identification of p53 gene structural changes in cancer cells can be facilitated through the application of a diverse series of high resolution, high throughput microarray platforms. Essentially two types of array include those that carry PCR products from cloned nucleic acids (e.g. cDNA, BACs, cosmids) and those that use oligonucleotides. The methods can provide a way to survey genome wide DNA copy number abnormalities and expression levels to allow correlations between losses, gains and amplifications in cancer cells with genes that are over- and under-expressed in the same samples. The gene expression arrays that provide estimates of mRNA levels in cancer cells have given rise to exon-specific arrays that can identify both gene expression levels, alternative splicing events and mRNA processing alterations.

Oligonucleotide arrays can be used to interrogate single nucleotide polymorphisms (SNPs) throughout the genome for linkage and association studies and these have been adapted to quantify copy number abnormalities and loss of heterozygosity events. DNA sequencing arrays can allow resequencing of chromosome regions, exomes, and whole genomes.

SNP-based arrays or other gene arrays or chips can determine the presence of wild type p53 allele and the structure of mutations. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome. For example, there are an estimated 5-10 million SNPs in the human genome. SNPs can be synonymous or nonsynonymous substitutions. Synonymous SNP substitutions do not result in a change of amino acid in the protein due to the degeneracy of the genetic code, but can affect function in other ways. For example, a seemingly silent mutation in gene that codes for a membrane transport protein can slow down translation, allowing the peptide chain to misfold, and produce a less functional mutant membrane transport protein. Nonsynonymous SNP substitutions can be missense substitutions or nonsense substitutions. Missense substitutions occur when a single base change results in change in amino acid sequence of the protein and malfunction thereof leads to disease. Nonsense substitutions occur when a point mutation results in a premature stop codon, or a nonsense codon in the transcribed mRNA, which results in a truncated and usually, nonfunctional, protein product. As SNPs are highly conserved throughout evolution and within a population, the map of SNPs serves as an excellent genotypic marker for research. SNP array is a useful tool to study the whole genome.

In addition, SNP array can be used for studying the Loss Of Heterozygosity (LOH). LOH is a form of allelic imbalance that can result from the complete loss of an allele or from an increase in copy number of one allele relative to the other. While other chip-based methods (e.g., comparative genomic hybridization can detect only genomic gains or deletions), SNP array has the additional advantage of detecting copy number neutral LOH due to uniparental disomy (UPD). In UPD, one allele or whole chromosome from one parent are missing leading to reduplication of the other parental allele (uni-parental=from one parent, disomy=duplicated). In a disease setting this occurrence can be pathologic when the wild type allele (e.g., from the mother) is missing and instead two copies of the heterozygous allele (e.g., from the father) are present. This usage of SNP array has a huge potential in cancer diagnostics as LOH is a prominent characteristic of most human cancers. SNP array technology have shown that cancers (e.g. gastric cancer, liver cancer, etc.) and hematologic malignancies (ALL, MDS, CML etc) have a high rate of LOH due to genomic deletions or UPD and genomic gains. In the present disclosure, using high density SNP array to detect LOH allows identification of pattern of allelic imbalance to determine the presence of wild type p53 allele (Lips et al., 2005; Lai et al., 2007).

Examples of p53 gene sequence and single nucleotide polymorphism arrays include p53 Gene Chip (Affymetrix, Santa Clara, Calif.), Roche p53 Ampli-Chip (Roche Molecular Systems, Pleasanton, Calif.), GeneChip Mapping arrays (Affymetrix, Santa Clara, Calif.), SNP Array 6.0 (Affymetrix, Santa Clara, Calif.), BeadArrays (Illumina, San Diego, Calif.), etc.

Mutations of wild type p53 genes can also be detected on the basis of the mutation of a wild type expression product of the p53 gene. Such expression products include both the mRNA as well as the p53 protein product itself. Point mutations can be detected by sequencing the mRNA directly or via molecular cloning of cDNA made from the mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques. The cDNA can also be sequenced via the polymerase chain reaction (PCR). A panel of monoclonal antibodies could be used in which each of the epitopes involved in p53 functions are represented by a monoclonal antibody. Loss or perturbation of binding of a monoclonal antibody in the panel can indicate mutational alteration of the p53 protein and thus of the p53 gene itself. Mutant p53 genes or gene products can also be detected in body samples, including, for example, serum, stool, urine, and sputum. The same techniques discussed above for detection of mutant p53 genes or gene products in tissues can be applied to other body samples.

Loss of wild type p53 genes can also be detected by screening for loss of wild type p53 protein function. Although all of the functions which the p53 protein undoubtedly possesses have yet to be elucidated, at least two specific functions are known. Protein p53 binds to the SV40 large T antigen as well as to the adenovirus E1B antigen. Loss of the ability of the p53 protein to bind to either or both of these antigens indicates a mutational alteration in the protein which reflects a mutational alteration of the gene itself. Alternatively, a panel of monoclonal antibodies could be used in which each of the epitopes involved in p53 functions are represented by a monoclonal antibody. Loss or perturbation of binding of a monoclonal antibody in the panel would indicate mutational alteration of the p53 protein and thus of the p53 gene itself. Any method for detecting an altered p53 protein can be used to detect loss of wild type p53 genes.

EXAMPLES Example 1: Peptidomimetic Macrocycles

Peptidomimetic macrocycles were synthesized, purified and analyzed as previously described and as described below (Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); and U.S. Pat. No. 7,192,713). Peptidomimetic macrocycles were designed by replacing two or more naturally occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at i and i+4, and i and i+7 positions. Peptide synthesis was performed either manually or on an automated peptide synthesizer (Applied Biosystems, model 433A), using solid phase conditions, rink amide AM resin (Novabiochem), and Fmoc main-chain protecting group chemistry. For the coupling of natural Fmoc-protected amino acids (Novabiochem), 10 equivalents of amino acid and a 1:1:2 molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4 equiv) were coupled with a 1:1:2 molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic peptides were acetylated, while the C-termini were amidated.

Purification of cross-linked compounds was achieved by high performance liquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18 column (Varian) to yield the pure compounds. Chemical composition of the pure products was confirmed by LC/MS mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC system) and amino acid analysis (Applied Biosystems, model 420A).

The following protocol was used in the synthesis of dialkyne-crosslinked peptidomimetic macrocycles, including SP662, SP663 and SP664. Fully protected resin-bound peptides were synthesized on a PEG-PS resin (loading 0.45 mmol/g) on a 0.2 mmol scale. Deprotection of the temporary Fmoc group was achieved by 3×10 min treatments of the resin bound peptide with 20% (v/v) piperidine in DMF. After washing with NMP (3×), dichloromethane (3×) and NMP (3×), coupling of each successive amino acid was achieved with 1×60 min incubation with the appropriate preactivated Fmoc-amino acid derivative. All protected amino acids (0.4 mmol) were dissolved in NMP and activated with HCTU (0.4 mmol) and DIEA (0.8 mmol) prior to transfer of the coupling solution to the deprotected resin-bound peptide. After coupling was completed, the resin was washed in preparation for the next deprotection/coupling cycle. Acetylation of the amino terminus was carried out in the presence of acetic anhydride/DIEA in NMP. The LC-MS analysis of a cleaved and deprotected sample obtained from an aliquot of the fully assembled resin-bound peptide was accomplished in order to verifying the completion of each coupling. In a typical example, tetrahydrofuran (4 ml) and triethylamine (2 ml) were added to the peptide resin (0.2 mmol) in a 40 ml glass vial and shaken for 10 minutes. Pd(PPh₃)₂Cl₂ (0.014 g, 0.02 mmol) and copper iodide (0.008 g, 0.04 mmol) were then added and the resulting reaction mixture was mechanically shaken 16 hours while open to atmosphere. The diyne-cyclized resin-bound peptides were deprotected and cleaved from the solid support by treatment with TFA/H₂O/TIS (95/5/5 v/v) for 2.5 h at room temperature. After filtration of the resin the TFA solution was precipitated in cold diethyl ether and centrifuged to yield the desired product as a solid. The crude product was purified by preparative HPLC.

The following protocol was used in the synthesis of single alkyne-crosslinked peptidomimetic macrocycles, including SP665. Fully protected resin-bound peptides were synthesized on a Rink amide MBHA resin (loading 0.62 mmol/g) on a 0.1 mmol scale. Deprotection of the temporary Fmoc group was achieved by 2×20 min treatments of the resin bound peptide with 25% (v/v) piperidine in NMP. After extensive flow washing with NMP and dichloromethane, coupling of each successive amino acid was achieved with 1×60 min incubation with the appropriate preactivated Fmoc-amino acid derivative. All protected amino acids (1 mmol) were dissolved in NMP and activated with HCTU (1 mmol) and DIEA (1 mmol) prior to transfer of the coupling solution to the deprotected resin-bound peptide. After coupling was completed, the resin was extensively flow washed in preparation for the next deprotection/coupling cycle. Acetylation of the amino terminus was carried out in the presence of acetic anhydride/DIEA in NMP/NMM. The LC-MS analysis of a cleaved and deprotected sample obtained from an aliquot of the fully assembled resin-bound peptide was accomplished in order to verifying the completion of each coupling. In a typical example, the peptide resin (0.1 mmol) was washed with DCM. Resin was loaded into a microwave vial. The vessel was evacuated and purged with nitrogen. Molybdenumhexacarbonyl (0.01 eq, Sigma Aldrich 199959) was added. Anhydrous chlorobenzene was added to the reaction vessel. Then 2-fluorophenol (leq, Sigma Aldrich F12804) was added. The reaction was then loaded into the microwave and held at 130° C. for 10 minutes. Reaction can need to be pushed a subsequent time for completion. The alkyne metathesized resin-bound peptides were deprotected and cleaved from the solid support by treatment with TFA/H₂O/TIS (94/3/3 v/v) for 3 h at room temperature. After filtration of the resin the TFA solution was precipitated in cold diethyl ether and centrifuged to yield the desired product as a solid. The crude product was purified by preparative HPLC.

Table 3 shows a list of peptidomimetic macrocycles prepared.

TABLE 3 SEQ ID Exact Found Calc Calc Calc SP Sequence NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP1 Ac-F$r8AYWEAc3cL$AAA-NH2 10 1456.78 729.44 1457.79 729.4 486.6 SP2 Ac-F$r8AYWEAc3cL$AAibA-NH2 11 1470.79 736.4 1471.8 736.4 491.27 SP3 Ac-LTF$r8AYWAQL$SANle-NH2 12 1715.97 859.02 1716.98 858.99 573 SP4 Ac-LTF$r8AYWAQL$SAL-NH2 13 1715.97 859.02 1716.98 858.99 573 SP5 Ac-LTF$r8AYWAQL$SAM-NH2 14 1733.92 868.48 1734.93 867.97 578.98 SP6 Ac-LTF$r8AYWAQL$SAhL-NH2 15 1729.98 865.98 1730.99 866 577.67 SP7 Ac-LTF$r8AYWAQL$SAF-NH2 16 1749.95 876.36 1750.96 875.98 584.32 SP8 Ac-LTF$r8AYWAQL$SAI-NH2 17 1715.97 859.02 1716.98 858.99 573 SP9 Ac-LTF$r8AYWAQL$SAChg-NH2 18 1741.98 871.98 1742.99 872 581.67 SP10 Ac-LTF$r8AYWAQL$SAAib-NH2 19 1687.93 845.36 1688.94 844.97 563.65 SP11 Ac-LTF$r8AYWAQL$SAA-NH2 20 1673.92 838.01 1674.93 837.97 558.98 SP12 Ac-LTF$r8AYWA$L$S$Nle-NH2 21 1767.04 884.77 1768.05 884.53 590.02 SP13 Ac-LTF$r8AYWA$L$S$A-NH2 22 1724.99 864.23 1726 863.5 576 SP14 Ac-F$r8AYWEAc3cL$AANle-NH2 23 1498.82 750.46 1499.83 750.42 500.61 SP15 Ac-F$r8AYWEAc3cL$AAL-NH2 24 1498.82 750.46 1499.83 750.42 500.61 SP16 Ac-F$r8AYWEAc3cL$AAM-NH2 25 1516.78 759.41 1517.79 759.4 506.6 SP17 Ac-F$r8AYWEAc3cL$AAhL-NH2 26 1512.84 757.49 1513.85 757.43 505.29 SP18 Ac-F$r8AYWEAc3cL$AAF-NH2 27 1532.81 767.48 1533.82 767.41 511.94 SP19 Ac-F$r8AYWEAc3cL$AAI-NH2 28 1498.82 750.39 1499.83 750.42 500.61 SP20 Ac-F$r8AYWEAc3cL$AAChg-NH2 29 1524.84 763.48 1525.85 763.43 509.29 SP21 Ac-F$r8AYWEAc3cL$AACha-NH2 30 1538.85 770.44 1539.86 770.43 513.96 SP22 Ac-F$r8AYWEAc3cL$AAAib-NH2 31 1470.79 736.84 1471.8 736.4 491.27 SP23 Ac-LTF$r8AYWAQL$AAAibV-NH2 32 1771.01 885.81 1772.02 886.51 591.34 SP24 Ac-LTF$r8AYWAQL$AAAibV-NH2 33 iso2 1771.01 886.26 1772.02 886.51 591.34 SP25 Ac-LTF$r8AYWAQL$SAibAA-NH2 34 1758.97 879.89 1759.98 880.49 587.33 SP26 Ac-LTF$r8AYWAQL$SAibAA-NH2 35 iso2 1758.97 880.34 1759.98 880.49 587.33 SP27 Ac-HLTF$r8HHWHQL$AANleNle-NH2 36 2056.15 1028.86 2057.16 1029.08 686.39 SP28 Ac-DLTF$r8HHWHQL$RRLV-NH2 37 2190.23 731.15 2191.24 1096.12 731.08 SP29 Ac-HHTF$r8HHWHQL$AAML-NH2 38 2098.08 700.43 2099.09 1050.05 700.37 SP30 Ac-F$r8HHWHQL$RRDCha-NH2 39 1917.06 959.96 1918.07 959.54 640.03 SP31 Ac-F$r8HHWHQL$HRFV-NH2 40 1876.02 938.65 1877.03 939.02 626.35 SP32 Ac-HLTF$r8HHWHQL$AAhLA-NH2 41 2028.12 677.2 2029.13 1015.07 677.05 SP33 Ac-DLTF$r8HHWHQL$RRChgl-NH2 42 2230.26 1115.89 2231.27 1116.14 744.43 SP34 Ac-DLTF$r8HHWHQL$RRChgl-NH2 43 iso2 2230.26 1115.96 2231.27 1116.14 744.43 SP35 Ac-HHTF$r8HHWHQL$AAChav-NH2 44 2106.14 1053.95 2107.15 1054.08 703.05 SP36 Ac-F$r8HHWHQL$RRDa-NH2 45 1834.99 918.3 1836 918.5 612.67 SP37 Ac-F$r8HHWHQL$HRAibG-NH2 46 1771.95 886.77 1772.96 886.98 591.66 SP38 Ac-F$r8AYWAQL$HHNleL-NH2 47 1730.97 866.57 1731.98 866.49 578 SP39 Ac-F$r8AYWSAL$HQANle-NH2 48 1638.89 820.54 1639.9 820.45 547.3 SP40 Ac-F$r8AYWVQL$QHChgl-NH2 49 1776.01 889.44 1777.02 889.01 593.01 SP41 Ac-F$r8AYWTAL$QQNlev-NH2 50 1671.94 836.97 1672.95 836.98 558.32 SP42 Ac-F$r8AYWYQL$HAibAa-NH2 51 1686.89 844.52 1687.9 844.45 563.3 SP43 Ac-LTF$r8AYWAQL$HHLa-NH2 52 1903.05 952.27 1904.06 952.53 635.36 SP44 Ac-LTF$r8AYWAQL$HHLa-NH2 53 iso2 1903.05 952.27 1904.06 952.53 635.36 SP45 Ac-LTF$r8AYWAQL$HQNlev-NH2 54 1922.08 962.48 1923.09 962.05 641.7 SP46 Ac-LTF$r8AYWAQL$HQNlev-NH2 55 iso2 1922.08 962.4 1923.09 962.05 641.7 SP47 Ac-LTF$r8AYWAQL$QQMl-NH2 56 1945.05 973.95 1946.06 973.53 649.36 SP48 Ac-LTF$r8AYWAQL$QQMl-NH2 57 iso2 1945.05 973.88 1946.06 973.53 649.36 SP49 Ac-LTF$r8AYWAQL$HAibhLV-NH2 58 1893.09 948.31 1894.1 947.55 632.04 SP50 Ac-LTF$r8AYWAQL$AHFA-NH2 59 1871.01 937.4 1872.02 936.51 624.68 SP51 Ac-HLTF$r8HHWHQL$AANlel-NH2 60 2056.15 1028.79 2057.16 1029.08 686.39 SP52 Ac-DLTF$r8HHWHQL$RRLa-NH2 61 2162.2 721.82 2163.21 1082.11 721.74 SP53 Ac-HHTF$r8HHWHQL$AAMv-NH2 62 2084.07 1042.92 2085.08 1043.04 695.7 SP54 Ac-F$r8HHWHQL$RRDA-NH2 63 1834.99 612.74 1836 918.5 612.67 SP55 Ac-F$r8HHWHQL$HRFCha-NH2 64 1930.06 966.47 1931.07 966.04 644.36 SP56 Ac-F$r8AYWEAL$AA-NHAm 65 1443.82 1445.71 1444.83 722.92 482.28 SP57 Ac-F$r8AYWEAL$AA-NHiAm 66 1443.82 723.13 1444.83 722.92 482.28 SP58 Ac-F$r8AYWEAL$AA-NHnPr3Ph 67 1491.82 747.3 1492.83 746.92 498.28 SP59 Ac-F$r8AYWEAL$AA-NHnBu33Me 68 1457.83 1458.94 1458.84 729.92 486.95 SP60 Ac-F$r8AYWEAL$AA-NHnPr 69 1415.79 709.28 1416.8 708.9 472.94 SP61 Ac-F$r8AYWEAL$AA-NHnEt2Ch 70 1483.85 1485.77 1484.86 742.93 495.62 SP62 Ac-F$r8AYWEAL$AA-NHnEt2Cp 71 1469.83 1470.78 1470.84 735.92 490.95 SP63 Ac-F$r8AYWEAL$AA-NHHex 72 1457.83 730.19 1458.84 729.92 486.95 SP64 Ac-LTF$r8AYWAQL$AAIA-NH2 73 1771.01 885.81 1772.02 886.51 591.34 SP65 Ac-LTF$r8AYWAQL$AAIA-NH2 74 iso2 1771.01 866.8 1772.02 886.51 591.34 SP66 Ac-LTF$r8AYWAAL$AAMA-NH2 75 1731.94 867.08 1732.95 866.98 578.32 SP67 Ac-LTF$r8AYWAAL$AAMA-NH2 76 iso2 1731.94 867.28 1732.95 866.98 578.32 SP68 Ac-LTF$r8AYWAQL$AANleA-NH2 77 1771.01 867.1 1772.02 886.51 591.34 SP69 Ac-LTF$r8AYWAQL$AANleA-NH2 78 iso2 1771.01 886.89 1772.02 886.51 591.34 SP70 Ac-LTF$r8AYWAQL$AAIa-NH2 79 1771.01 886.8 1772.02 886.51 591.34 SP71 Ac-LTF$r8AYWAQL$AAIa-NH2 80 iso2 1771.01 887.09 1772.02 886.51 591.34 SP72 Ac-LTF$r8AYWAAL$AAMa-NH2 81 1731.94 867.17 1732.95 866.98 578.32 SP73 Ac-LTF$r8AYWAAL$AAMa-NH2 82 iso2 1731.94 867.37 1732.95 866.98 578.32 SP74 Ac-LTF$r8AYWAQL$AANlea-NH2 83 1771.01 887.08 1772.02 886.51 591.34 SP75 Ac-LTF$r8AYWAQL$AANlea-NH2 84 iso2 1771.01 887.08 1772.02 886.51 591.34 SP76 Ac-LTF$r8AYWAAL$AAIv-NH2 85 1742.02 872.37 1743.03 872.02 581.68 SP77 Ac-LTF$r8AYWAAL$AAIv-NH2 86 iso2 1742.02 872.74 1743.03 872.02 581.68 SP78 Ac-LTF$r8AYWAQL$AAMv-NH2 87 1817 910.02 1818.01 909.51 606.67 SP79 Ac-LTF$r8AYWAAL$AANlev-NH2 88 1742.02 872.37 1743.03 872.02 581.68 SP80 Ac-LTF$r8AYWAAL$AANlev-NH2 89 iso2 1742.02 872.28 1743.03 872.02 581.68 SP81 Ac-LTF$r8AYWAQL$AAIl-NH2 90 1813.05 907.81 1814.06 907.53 605.36 SP82 Ac-LTF$r8AYWAQL$AAIl-NH2 91 iso2 1813.05 907.81 1814.06 907.53 605.36 SP83 Ac-LTF$r8AYWAAL$AAMl-NH2 92 1773.99 887.37 1775 888 592.34 SP84 Ac-LTF$r8AYWAQL$AANlel-NH2 93 1813.05 907.61 1814.06 907.53 605.36 SP85 Ac-LTF$r8AYWAQL$AANlel-NH2 94 iso2 1813.05 907.71 1814.06 907.53 605.36 SP86 Ac-F$r8AYWEAL$AAMA-NH2 95 1575.82 789.02 1576.83 788.92 526.28 SP87 Ac-F$r8AYWEAL$AANleA-NH2 96 1557.86 780.14 1558.87 779.94 520.29 SP88 Ac-F$r8AYWEAL$AAIa-NH2 97 1557.86 780.33 1558.87 779.94 520.29 SP89 Ac-F$r8AYWEAL$AAMa-NH2 98 1575.82 789.3 1576.83 788.92 526.28 SP90 Ac-F$r8AYWEAL$AANlea-NH2 99 1557.86 779.4 1558.87 779.94 520.29 SP91 Ac-F$r8AYWEAL$AAIv-NH2 100 1585.89 794.29 1586.9 793.95 529.64 SP92 Ac-F$r8AYWEAL$AAMv-NH2 101 1603.85 803.08 1604.86 802.93 535.62 SP93 Ac-F$r8AYWEAL$AANlev-NH2 102 1585.89 793.46 1586.9 793.95 529.64 SP94 Ac-F$r8AYWEAL$AAIl-NH2 103 1599.91 800.49 1600.92 800.96 534.31 SP95 Ac-F$r8AYWEAL$AAMl-NH2 104 1617.86 809.44 1618.87 809.94 540.29 SP96 Ac-F$r8AYWEAL$AANlel-NH2 105 1599.91 801.7 1600.92 800.96 534.31 SP97 Ac-F$r8AYWEAL$AANlel-NH2 106 iso2 1599.91 801.42 1600.92 800.96 534.31 SP98 Ac-LTF$r8AY6clWAQL$SAA-NH2 107 1707.88 855.72 1708.89 854.95 570.3 SP99 Ac-LTF$r8AY6clWAQL$SAA-NH2 108 iso2 1707.88 855.35 1708.89 854.95 570.3 SP100 Ac-WTF$r8FYWSQL$AVAa-NH2 109 1922.01 962.21 1923.02 962.01 641.68 SP101 Ac-WTF$r8FYWSQL$AVAa-NH2 110 iso2 1922.01 962.49 1923.02 962.01 641.68 SP102 Ac-WTF$r8VYWSQL$AVA-NH2 111 1802.98 902.72 1803.99 902.5 602 SP103 Ac-WTF$r8VYWSQL$AVA-NH2 112 iso2 1802.98 903 1803.99 902.5 602 SP104 Ac-WTF$r8FYWSQL$SAAa-NH2 113 1909.98 956.47 1910.99 956 637.67 SP105 Ac-WTF$r8FYWSQL$SAAa-NH2 114 iso2 1909.98 956.47 1910.99 956 637.67 SP106 Ac-WTF$r8VYWSQL$AVAaa-NH2 115 1945.05 974.15 1946.06 973.53 649.36 SP107 Ac-WTF$r8VYWSQL$AVAaa-NH2 116 iso2 1945.05 973.78 1946.06 973.53 649.36 SP108 Ac-LTF$r8AYWAQL$AVG-NH2 117 1671.94 837.52 1672.95 836.98 558.32 SP109 Ac-LTF$r8AYWAQL$AVG-NH2 118 iso2 1671.94 837.21 1672.95 836.98 558.32 SP110 Ac-LTF$r8AYWAQL$AVQ-NH2 119 1742.98 872.74 1743.99 872.5 582 SP111 Ac-LTF$r8AYWAQL$AVQ-NH2 120 iso2 1742.98 872.74 1743.99 872.5 582 SP112 Ac-LTF$r8AYWAQL$SAa-NH2 121 1673.92 838.23 1674.93 837.97 558.98 SP113 Ac-LTF$r8AYWAQL$SAa-NH2 122 iso2 1673.92 838.32 1674.93 837.97 558.98 SP114 Ac-LTF$r8AYWAQhL$SAA-NH2 123 1687.93 844.37 1688.94 844.97 563.65 SP115 Ac-LTF$r8AYWAQhL$SAA-NH2 124 iso2 1687.93 844.81 1688.94 844.97 563.65 SP116 Ac-LTF$r8AYWEQLStSA$-NH2 125 1826 905.27 1827.01 914.01 609.67 SP117 Ac-LTF$r8AYWAQL$SLA-NH2 126 1715.97 858.48 1716.98 858.99 573 SP118 Ac-LTF$r8AYWAQL$SLA-NH2 127 iso2 1715.97 858.87 1716.98 858.99 573 SP119 Ac-LTF$r8AYWAQL$SWA-NH2 128 1788.96 895.21 1789.97 895.49 597.33 SP120 Ac-LTF$r8AYWAQL$SWA-NH2 129 iso2 1788.96 895.28 1789.97 895.49 597.33 SP121 Ac-LTF$r8AYWAQL$SVS-NH2 130 1717.94 859.84 1718.95 859.98 573.65 SP122 Ac-LTF$r8AYWAQL$SAS-NH2 131 1689.91 845.85 1690.92 845.96 564.31 SP123 Ac-LTF$r8AYWAQL$SVG-NH2 132 1687.93 844.81 1688.94 844.97 563.65 SP124 Ac-ETF$r8VYWAQL$SAa-NH2 133 1717.91 859.76 1718.92 859.96 573.64 SP125 Ac-ETF$r8VYWAQL$SAA-NH2 134 1717.91 859.84 1718.92 859.96 573.64 SP126 Ac-ETF$r8VYWAQL$SVA-NH2 135 1745.94 873.82 1746.95 873.98 582.99 SP127 Ac-ETF$r8VYWAQL$SLA-NH2 136 1759.96 880.85 1760.97 880.99 587.66 SP128 Ac-ETF$r8VYWAQL$SWA-NH2 137 1832.95 917.34 1833.96 917.48 611.99 SP129 Ac-ETF$r8KYWAQL$SWA-NH2 138 1861.98 931.92 1862.99 932 621.67 SP130 Ac-ETF$r8VYWAQL$SVS-NH2 139 1761.93 881.89 1762.94 881.97 588.32 SP131 Ac-ETF$r8VYWAQL$SAS-NH2 140 1733.9 867.83 1734.91 867.96 578.97 SP132 Ac-ETF$r8VYWAQL$SVG-NH2 141 1731.92 866.87 1732.93 866.97 578.31 SP133 Ac-LTF$r8VYWAQL$SSa-NH2 142 1717.94 859.47 1718.95 859.98 573.65 SP134 Ac-ETF$r8VYWAQL$SSa-NH2 143 1733.9 867.83 1734.91 867.96 578.97 SP135 Ac-LTF$r8VYWAQL$SNa-NH2 144 1744.96 873.38 1745.97 873.49 582.66 SP136 Ac-ETF$r8VYWAQL$SNa-NH2 145 1760.91 881.3 1761.92 881.46 587.98 SP137 Ac-LTF$r8VYWAQL$SAa-NH2 146 1701.95 851.84 1702.96 851.98 568.32 SP138 Ac-LTF$r8VYWAQL$SVA-NH2 147 1729.98 865.53 1730.99 866 577.67 SP139 Ac-LTF$r8VYWAQL$SVA-NH2 148 iso2 1729.98 865.9 1730.99 866 577.67 SP140 Ac-LTF$r8VYWAQL$SWA-NH2 149 1816.99 909.42 1818 909.5 606.67 SP141 Ac-LTF$r8VYWAQL$SVS-NH2 150 1745.98 873.9 1746.99 874 583 SP142 Ac-LTF$r8VYWAQL$SVS-NH2 151 iso2 1745.98 873.9 1746.99 874 583 SP143 Ac-LTF$r8VYWAQL$SAS-NH2 152 1717.94 859.84 1718.95 859.98 573.65 SP144 Ac-LTF$r8VYWAQL$SAS-NH2 153 iso2 1717.94 859.91 1718.95 859.98 573.65 SP145 Ac-LTF$r8VYWAQL$SVG-NH2 154 1715.97 858.87 1716.98 858.99 573 SP146 Ac-LTF$r8VYWAQL$SVG-NH2 155 iso2 1715.97 858.87 1716.98 858.99 573 SP147 Ac-LTF$r8EYWAQCha$SAA-NH2 156 1771.96 886.85 1772.97 886.99 591.66 SP148 Ac-LTF$r8EYWAQCha$SAA-NH2 157 iso2 1771.96 886.85 1772.97 886.99 591.66 SP149 Ac-LTF$r8EYWAQCpg$SAA-NH2 158 1743.92 872.86 1744.93 872.97 582.31 SP150 Ac-LTF$r8EYWAQCpg$SAA-NH2 159 iso2 1743.92 872.86 1744.93 872.97 582.31 SP151 Ac-LTF$r8EYWAQF$SAA-NH2 160 1765.91 883.44 1766.92 883.96 589.64 SP152 Ac-LTF$r8EYWAQF$SAA-NH2 161 iso2 1765.91 883.89 1766.92 883.96 589.64 SP153 Ac-LTF$r8EYWAQCba$SAA-NH2 162 1743.92 872.42 1744.93 872.97 582.31 SP154 Ac-LTF$r8EYWAQCba$SAA-NH2 163 iso2 1743.92 873.39 1744.93 872.97 582.31 SP155 Ac-LTF3Cl$r8EYWAQL$SAA-NH2 164 1765.89 883.89 1766.9 883.95 589.64 SP156 Ac-LTF3Cl$r8EYWAQL$SAA-NH2 165 iso2 1765.89 883.96 1766.9 883.95 589.64 SP157 Ac-LTF34F2$r8EYWAQL$SAA-NH2 166 1767.91 884.48 1768.92 884.96 590.31 SP158 Ac-LTF34F2$r8EYWAQL$SAA-NH2 167 iso2 1767.91 884.48 1768.92 884.96 590.31 SP159 Ac-LTF34F2$r8EYWAQhL$SAA-NH2 168 1781.92 891.44 1782.93 891.97 594.98 SP160 Ac-LTF34F2$r8EYWAQhL$SAA-NH2 169 iso2 1781.92 891.88 1782.93 891.97 594.98 SP161 Ac-ETF$r8EYWAQL$SAA-NH2 170 1747.88 874.34 1748.89 874.95 583.63 SP162 Ac-LTF$r8AYWVQL$SAA-NH2 171 1701.95 851.4 1702.96 851.98 568.32 SP163 Ac-LTF$r8AHWAQL$SAA-NH2 172 1647.91 824.83 1648.92 824.96 550.31 SP164 Ac-LTF$r8AEWAQL$SAA-NH2 173 1639.9 820.39 1640.91 820.96 547.64 SP165 Ac-LTF$r8ASWAQL$SAA-NH2 174 1597.89 799.38 1598.9 799.95 533.64 SP166 Ac-LTF$r8AEWAQL$SAA-NH2 175 iso2 1639.9 820.39 1640.91 820.96 547.64 SP167 Ac-LTF$r8ASWAQL$SAA-NH2 176 iso2 1597.89 800.31 1598.9 799.95 533.64 SP168 Ac-LTF$r8AF4coohWAQL$SAA-NH2 177 1701.91 851.4 1702.92 851.96 568.31 SP169 Ac-LTF$r8AF4coohWAQL$SAA-NH2 178 iso2 1701.91 851.4 1702.92 851.96 568.31 SP170 Ac-LTF$r8AHWAQL$AAIa-NH2 179 1745 874.13 1746.01 873.51 582.67 SP171 Ac-ITF$r8FYWAQL$AAIa-NH2 180 1847.04 923.92 1848.05 924.53 616.69 SP172 Ac-ITF$r8EHWAQL$AAIa-NH2 181 1803.01 903.17 1804.02 902.51 602.01 SP173 Ac-ITF$r8EHWAQL$AAIa-NH2 182 iso2 1803.01 903.17 1804.02 902.51 602.01 SP174 Ac-ETF$r8EHWAQL$AAIa-NH2 183 1818.97 910.76 1819.98 910.49 607.33 SP175 Ac-ETF$r8EHWAQL$AAIa-NH2 184 iso2 1818.97 910.85 1819.98 910.49 607.33 SP176 Ac-LTF$r8AHWVQL$AAIa-NH2 185 1773.03 888.09 1774.04 887.52 592.02 SP177 Ac-ITF$r8FYWVQL$AAIa-NH2 186 1875.07 939.16 1876.08 938.54 626.03 SP178 Ac-ITF$r8EYWVQL$AAIa-NH2 187 1857.04 929.83 1858.05 929.53 620.02 SP179 Ac-ITF$r8EHWVQL$AAIa-NH2 188 1831.04 916.86 1832.05 916.53 611.35 SP180 Ac-LTF$r8AEWAQL$AAIa-NH2 189 1736.99 869.87 1738 869.5 580 SP181 Ac-LTF$r8AF4coohWAQL$AAIa-NH2 190 1799 900.17 1800.01 900.51 600.67 SP182 Ac-LTF$r8AF4coohWAQL$AAIa-NH2 191 iso2 1799 900.24 1800.01 900.51 600.67 SP183 Ac-LTF$r8AHWAQL$AHFA-NH2 192 1845.01 923.89 1846.02 923.51 616.01 SP184 Ac-ITF$r8FYWAQUARFA-NH2 193 1947.05 975.05 1948.06 974.53 650.02 SP185 Ac-ITF$r8FYWAQUARFA-NH2 194 iso2 1947.05 976.07 1948.06 974.53 650.02 SP186 Ac-ITF$r8FHWAQL$AEFA-NH2 195 1913.02 958.12 1914.03 957.52 638.68 SP187 Ac-ITF$r8FHWAQL$AEFA-NH2 196 iso2 1913.02 957.86 1914.03 957.52 638.68 SP188 Ac-ITF$r8EHWAQL$AHFA-NH2 197 1903.01 952.94 1904.02 952.51 635.34 SP189 Ac-ITF$r8EHWAQL$AHFA-NH2 198 iso2 1903.01 953.87 1904.02 952.51 635.34 SP190 Ac-LTF$r8AHWVQL$AHFA-NH2 199 1873.04 937.86 1874.05 937.53 625.35 SP191 Ac-ITF$r8FYWVQL$AHFA-NH2 200 1975.08 988.83 1976.09 988.55 659.37 SP192 Ac-ITF$r8EYWVQL$AHFA-NH2 201 1957.05 979.35 1958.06 979.53 653.36 SP193 Ac-ITF$r8EHWVQL$AHFA-NH2 202 1931.05 967 1932.06 966.53 644.69 SP194 Ac-ITF$r8EHWVQL$AHFA-NH2 203 iso2 1931.05 967.93 1932.06 966.53 644.69 SP195 Ac-ETF$r8EYWAAL$SAA-NH2 204 1690.86 845.85 1691.87 846.44 564.63 SP196 Ac-LTF$r8AYWVAL$SAA-NH2 205 1644.93 824.08 1645.94 823.47 549.32 SP197 Ac-LTF$r8AHWAAL$SAA-NH2 206 1590.89 796.88 1591.9 796.45 531.3 SP198 Ac-LTF$r8AEWAAL$SAA-NH2 207 1582.88 791.9 1583.89 792.45 528.63 SP199 Ac-LTF$r8AEWAAL$SAA-NH2 208 iso2 1582.88 791.9 1583.89 792.45 528.63 SP200 Ac-LTF$r8ASWAAL$SAA-NH2 209 1540.87 770.74 1541.88 771.44 514.63 SP201 Ac-LTF$r8ASWAAL$SAA-NH2 210 iso2 1540.87 770.88 1541.88 771.44 514.63 SP202 Ac-LTF$r8AYWAAL$AAIa-NH2 211 1713.99 857.39 1715 858 572.34 SP203 Ac-LTF$r8AYWAAL$AAIa-NH2 212 iso2 1713.99 857.84 1715 858 572.34 SP204 Ac-LTF$r8AYWAAL$AHFA-NH2 213 1813.99 907.86 1815 908 605.67 SP205 Ac-LTF$r8EHWAQL$AHIa-NH2 214 1869.03 936.1 1870.04 935.52 624.02 SP206 Ac-LTF$r8EHWAQL$AHIa-NH2 215 iso2 1869.03 937.03 1870.04 935.52 624.02 SP207 Ac-LTF$r8AHWAQL$AHIa-NH2 216 1811.03 906.87 1812.04 906.52 604.68 SP208 Ac-LTF$r8EYWAQL$AHIa-NH2 217 1895.04 949.15 1896.05 948.53 632.69 SP209 Ac-LTF$r8AYWAQL$AAFa-NH2 218 1804.99 903.2 1806 903.5 602.67 SP210 Ac-LTF$r8AYWAQL$AAFa-NH2 219 iso2 1804.99 903.28 1806 903.5 602.67 SP211 Ac-LTF$r8AYWAQL$AAWa-NH2 220 1844 922.81 1845.01 923.01 615.67 SP212 Ac-LTF$r8AYWAQL$AAVa-NH2 221 1756.99 878.86 1758 879.5 586.67 SP213 Ac-LTF$r8AYWAQL$AAVa-NH2 222 iso2 1756.99 879.3 1758 879.5 586.67 SP214 Ac-LTF$r8AYWAQL$AALa-NH2 223 1771.01 886.26 1772.02 886.51 591.34 SP215 Ac-LTF$r8AYWAQL$AALa-NH2 224 iso2 1771.01 886.33 1772.02 886.51 591.34 SP216 Ac-LTF$r8EYWAQL$AAIa-NH2 225 1829.01 914.89 1830.02 915.51 610.68 SP217 Ac-LTF$r8EYWAQL$AAIa-NH2 226 iso2 1829.01 915.34 1830.02 915.51 610.68 SP218 Ac-LTF$r8EYWAQL$AAFa-NH2 227 1863 932.87 1864.01 932.51 622.01 SP219 Ac-LTF$r8EYWAQL$AAFa-NH2 228 iso2 1863 932.87 1864.01 932.51 622.01 SP220 Ac-LTF$r8EYWAQL$AAVa-NH2 229 1815 908.23 1816.01 908.51 606.01 SP221 Ac-LTF$r8EYWAQL$AAVa-NH2 230 iso2 1815 908.31 1816.01 908.51 606.01 SP222 Ac-LTF$r8EHWAQL$AAIa-NH2 231 1803.01 903.17 1804.02 902.51 602.01 SP223 Ac-LTF$r8EHWAQL$AAIa-NH2 232 iso2 1803.01 902.8 1804.02 902.51 602.01 SP224 Ac-LTF$r8EHWAQL$AAWa-NH2 233 1876 939.34 1877.01 939.01 626.34 SP225 Ac-LTF$r8EHWAQL$AAWa-NH2 234 iso2 1876 939.62 1877.01 939.01 626.34 SP226 Ac-LTF$r8EHWAQL$AALa-NH2 235 1803.01 902.8 1804.02 902.51 602.01 SP227 Ac-LTF$r8EHWAQL$AALa-NH2 236 iso2 1803.01 902.9 1804.02 902.51 602.01 SP228 Ac-ETF$r8EHWVQL$AALa-NH2 237 1847 924.82 1848.01 924.51 616.67 SP229 Ac-LTF$r8AYWAQL$AAAa-NH2 238 1728.96 865.89 1729.97 865.49 577.33 SP230 Ac-LTF$r8AYWAQL$AAAa-NH2 239 iso2 1728.96 865.89 1729.97 865.49 577.33 SP231 Ac-LTF$r8AYWAQL$AAAibA-NH2 240 1742.98 872.83 1743.99 872.5 582 SP232 Ac-LTF$r8AYWAQL$AAAibA-NH2 241 iso2 1742.98 872.92 1743.99 872.5 582 SP233 Ac-LTF$r8AYWAQL$AAAAa-NH2 242 1800 901.42 1801.01 901.01 601.01 SP234 Ac-LTF$r5AYWAQL$s8AAIa-NH2 243 1771.01 887.17 1772.02 886.51 591.34 SP235 Ac-LTF$r5AYWAQL$s8SAA-NH2 244 1673.92 838.33 1674.93 837.97 558.98 SP236 Ac-LTF$r8AYWAQCba$AANleA-NH2 245 1783.01 892.64 1784.02 892.51 595.34 SP237 Ac-ETF$r8AYWAQCba$AANleA-NH2 246 1798.97 900.59 1799.98 900.49 600.66 SP238 Ac-LTF$r8EYWAQCba$AANleA-NH2 247 1841.01 922.05 1842.02 921.51 614.68 SP239 Ac-LTF$r8AYWAQCba$AWNleA-NH2 248 1898.05 950.46 1899.06 950.03 633.69 SP240 Ac-ETF$r8AYWAQCba$AWNleA-NH2 249 1914.01 958.11 1915.02 958.01 639.01 SP241 Ac-LTF$r8EYWAQCba$AWNleA-NH2 250 1956.06 950.62 1957.07 979.04 653.03 SP242 Ac-LTF$r8EYWAQCba$SAFA-NH2 251 1890.99 946.55 1892 946.5 631.34 SP243 Ac-LTF34F2$r8EYWAQCba$SANleA- 252 1892.99 947.57 1894 947.5 632 NH2 SP244 Ac-LTF$r8EF4coohWAQCba$SANleA- 253 1885 943.59 1886.01 943.51 629.34 NH2 SP245 Ac-LTF$r8EYWSQCba$SANleA-NH2 254 1873 937.58 1874.01 937.51 625.34 SP246 Ac-LTF$r8EYWWQCba$SANleA-NH2 255 1972.05 987.61 1973.06 987.03 658.36 SP247 Ac-LTF$r8EYWAQCba$AAIa-NH2 256 1841.01 922.05 1842.02 921.51 614.68 SP248 Ac-LTF34F2$r8EYWAQCba$AAIa-NH2 257 1876.99 939.99 1878 939.5 626.67 SP249 Ac-LTF$r8EF4coohWAQCba$AAIa-NH2 258 1869.01 935.64 1870.02 935.51 624.01 SP250 Pam-ETF$r8EYWAQCba$SAA-NH2 259 1956.1 979.57 1957.11 979.06 653.04 SP251 Ac-LThF$r8EFWAQCba$SAA-NH2 260 1741.94 872.11 1742.95 871.98 581.65 SP252 Ac-LTA$r8EYWAQCba$SAA-NH2 261 1667.89 835.4 1668.9 834.95 556.97 SP253 Ac-LTF$r8EYAAQCba$SAA-NH2 262 1628.88 815.61 1629.89 815.45 543.97 SP254 Ac-LTF$r8EY2NalAQCba$SAA-NH2 263 1754.93 879.04 1755.94 878.47 585.98 SP255 Ac-LTF$r8AYWAQCba$SAA-NH2 264 1685.92 844.71 1686.93 843.97 562.98 SP256 Ac-LTF$r8EYWAQCba$SAF-NH2 265 1819.96 911.41 1820.97 910.99 607.66 SP257 Ac-LTF$r8EYWAQCba$SAFa-NH2 266 1890.99 947.41 1892 946.5 631.34 SP258 Ac-LTF$r8AYWAQCba$SAF-NH2 267 1761.95 882.73 1762.96 881.98 588.32 SP259 Ac-LTF34F2$r8AYWAQCba$SAF-NH2 268 1797.93 900.87 1798.94 899.97 600.32 SP260 Ac-LTF$r8AF4coohWAQCba$SAF-NH2 269 1789.94 896.43 1790.95 895.98 597.65 SP261 Ac-LTF$r8EY6clWAQCba$SAF-NH2 270 1853.92 929.27 1854.93 927.97 618.98 SP262 Ac-LTF$r8AYWSQCba$SAF-NH2 271 1777.94 890.87 1778.95 889.98 593.65 SP263 Ac-LTF$r8AYWWQCba$SAF-NH2 272 1876.99 939.91 1878 939.5 626.67 SP264 Ac-LTF$r8AYWAQCba$AAIa-NH2 273 1783.01 893.19 1784.02 892.51 595.34 SP265 Ac-LTF34F2$r8AYWAQCba$AAIa-NH2 274 1818.99 911.23 1820 910.5 607.34 SP266 Ac-LTF$r8AY6clWAQCba$AAIa-NH2 275 1816.97 909.84 1817.98 909.49 606.66 SP267 Ac-LTF$r8AF4coohWAQCba$AAIa-NH2 276 1811 906.88 1812.01 906.51 604.67 SP268 Ac-LTF$r8EYWAQCba$AAFa-NH2 277 1875 938.6 1876.01 938.51 626.01 SP269 Ac-LTF$r8EYWAQCba$AAFa-NH2 278 iso2 1875 938.6 1876.01 938.51 626.01 SP270 Ac-ETF$r8AYWAQCba$AWNlea-NH2 279 1914.01 958.42 1915.02 958.01 639.01 SP271 Ac-LTF$r8EYWAQCba$AWNlea-NH2 280 1956.06 979.42 1957.07 979.04 653.03 SP272 Ac-ETF$r8EYWAQCba$AWNlea-NH2 281 1972.01 987.06 1973.02 987.01 658.34 SP273 Ac-ETF$r8EYWAQCba$AWNlea-NH2 282 iso2 1972.01 987.06 1973.02 987.01 658.34 SP274 Ac-LTF$r8AYWAQCba$SAFa-NH2 283 1832.99 917.89 1834 917.5 612 SP275 Ac-LTF$r8AYWAQCba$SAFa-NH2 284 iso2 1832.99 918.07 1834 917.5 612 SP276 Ac-ETF$r8AYWAQL$AWNlea-NH2 285 1902.01 952.22 1903.02 952.01 635.01 SP277 Ac-LTF$r8EYWAQL$AWNlea-NH2 286 1944.06 973.5 1945.07 973.04 649.03 SP278 Ac-ETF$r8EYWAQL$AWNlea-NH2 287 1960.01 981.46 1961.02 981.01 654.34 SP279 Dmaac-LTF$r8EYWAQhL$SAA-NH2 288 1788.98 896.06 1789.99 895.5 597.33 SP280 Hexac-LTF$r8EYWAQhL$SAA-NH2 289 1802 902.9 1803.01 902.01 601.67 SP281 Napac-LTF$r8EYWAQhL$SAA-NH2 290 1871.99 937.58 1873 937 625 SP282 Decac-LTF$r8EYWAQhL$SAA-NH2 291 1858.06 930.55 1859.07 930.04 620.36 SP283 Admac-LTF$r8EYWAQhL$SAA-NH2 292 1866.03 934.07 1867.04 934.02 623.02 SP284 Tmac-LTF$r8EYWAQhL$SAA-NH2 293 1787.99 895.41 1789 895 597 SP285 Pam-LTF$r8EYWAQhL$SAA-NH2 294 1942.16 972.08 1943.17 972.09 648.39 SP286 Ac-LTF$r8AYWAQCba$AANleA-NH2 295 iso2 1783.01 892.64 1784.02 892.51 595.34 SP287 Ac-LTF34F2$r8EYWAQCba$AAIa-NH2 296 iso2 1876.99 939.62 1878 939.5 626.67 SP288 Ac-LTF34F2$r8EYWAQCba$SAA-NH2 297 1779.91 892.07 1780.92 890.96 594.31 SP289 Ac-LTF34F2$r8EYWAQCba$SAA-NH2 298 iso2 1779.91 891.61 1780.92 890.96 594.31 SP290 Ac-LTF$r8EF4coohWAQCba$SAA-NH2 299 1771.92 887.54 1772.93 886.97 591.65 SP291 Ac-LTF$r8EF4coohWAQCba$SAA-NH2 300 iso2 1771.92 887.63 1772.93 886.97 591.65 SP292 Ac-LTF$r8EYWSQCba$SAA-NH2 301 1759.92 881.9 1760.93 880.97 587.65 SP293 Ac-LTF$r8EYWSQCba$SAA-NH2 302 iso2 1759.92 881.9 1760.93 880.97 587.65 SP294 Ac-LTF$r8EYWAQhL$SAA-NH2 303 1745.94 875.05 1746.95 873.98 582.99 SP295 Ac-LTF$r8AYWAQhL$SAF-NH2 304 1763.97 884.02 1764.98 882.99 589 SP296 Ac-LTF$r8AYWAQhL$SAF-NH2 305 iso2 1763.97 883.56 1764.98 882.99 589 SP297 Ac-LTF34F2$r8AYWAQhL$SAA-NH2 306 1723.92 863.67 1724.93 862.97 575.65 SP298 Ac-LTF34F2$r8AYWAQhL$SAA-NH2 307 iso2 1723.92 864.04 1724.93 862.97 575.65 SP299 Ac-LTF$r8AF4coohWAQhL$SAA-NH2 308 1715.93 859.44 1716.94 858.97 572.98 SP300 Ac-LTF$r8AF4coohWAQhL$SAA-NH2 309 iso2 1715.93 859.6 1716.94 858.97 572.98 SP301 Ac-LTF$r8AYWSQhL$SAA-NH2 310 1703.93 853.96 1704.94 852.97 568.98 SP302 Ac-LTF$r8AYWSQhL$SAA-NH2 311 iso2 1703.93 853.59 1704.94 852.97 568.98 SP303 Ac-LTF$r8EYWAQL$AANleA-NH2 312 1829.01 915.45 1830.02 915.51 610.68 SP304 Ac-LTF34F2$r8AYWAQL$AANleA-NH2 313 1806.99 904.58 1808 904.5 603.34 SP305 Ac-LTF$r8AF4coohWAQL$AANleA-NH2 314 1799 901.6 1800.01 900.51 600.67 SP306 Ac-LTF$r8AYWSQL$AANleA-NH2 315 1787 894.75 1788.01 894.51 596.67 SP307 Ac-LTF34F2$r8AYWAQhL$AANleA-NH2 316 1821 911.79 1822.01 911.51 608.01 SP308 Ac-LTF34F2$r8AYWAQhL$AANleA-NH2 317 iso2 1821 912.61 1822.01 911.51 608.01 SP309 Ac-LTF$r8AF4coohWAQhL$AANleA- 318 1813.02 907.95 1814.03 907.52 605.35 NH2 SP310 Ac-LTF$r8AF4coohWAQhL$AANleA- 319 iso2 1813.02 908.54 1814.03 907.52 605.35 NH2 SP311 Ac-LTF$r8AYWSQhL$AANleA-NH2 320 1801.02 901.84 1802.03 901.52 601.35 SP312 Ac-LTF$r8AYWSQhL$AANleA-NH2 321 iso2 1801.02 902.62 1802.03 901.52 601.35 SP313 Ac-LTF$r8AYWAQhL$AAAAa-NH2 322 1814.01 908.63 1815.02 908.01 605.68 SP314 Ac-LTF$r8AYWAQhL$AAAAa-NH2 323 iso2 1814.01 908.34 1815.02 908.01 605.68 SP315 Ac-LTF$r8AYWAQL$AAAAAa-NH2 324 1871.04 936.94 1872.05 936.53 624.69 SP316 Ac-LTF$r8AYWAQL$AAAAAAa-NH2 325 iso2 1942.07 972.5 1943.08 972.04 648.37 SP317 Ac-LTF$r8AYWAQL$AAAAAAa-NH2 326 iso1 1942.07 972.5 1943.08 972.04 648.37 SP318 Ac-LTF$r8EYWAQhL$AANleA-NH2 327 1843.03 922.54 1844.04 922.52 615.35 SP319 Ac-AATF$r8AYWAQL$AANleA-NH2 328 1800 901.39 1801.01 901.01 601.01 SP320 Ac-LTF$r8AYWAQL$AANleAA-NH2 329 1842.04 922.45 1843.05 922.03 615.02 SP321 Ac-ALTF$r8AYWAQL$AANleAA-NH2 330 1913.08 957.94 1914.09 957.55 638.7 SP322 Ac-LTF$r8AYWAQCba$AANleAA-NH2 331 1854.04 928.43 1855.05 928.03 619.02 SP323 Ac-LTF$r8AYWAQhL$AANleAA-NH2 332 1856.06 929.4 1857.07 929.04 619.69 SP324 Ac-LTF$r8EYWAQCba$SAAA-NH2 333 1814.96 909.37 1815.97 908.49 605.99 SP325 Ac-LTF$r8EYWAQCba$SAAA-NH2 334 iso2 1814.96 909.37 1815.97 908.49 605.99 SP326 Ac-LTF$r8EYWAQCba$SAAAA-NH2 335 1886 944.61 1887.01 944.01 629.67 SP327 Ac-LTF$r8EYWAQCba$SAAAA-NH2 336 iso2 1886 944.61 1887.01 944.01 629.67 SP328 Ac-ALTF$r8EYWAQCba$SAA-NH2 337 1814.96 909.09 1815.97 908.49 605.99 SP329 Ac-ALTF$r8EYWAQCba$SAAA-NH2 338 1886 944.61 1887.01 944.01 629.67 SP330 Ac-ALTF$r8EYWAQCba$SAA-NH2 339 iso2 1814.96 909.09 1815.97 908.49 605.99 SP331 Ac-LTF$r8EYWAQL$AAAAAa-NH2 340 iso2 1929.04 966.08 1930.05 965.53 644.02 SP332 Ac-LTF$r8EY6clWAQCba$SAA-NH2 341 1777.89 890.78 1778.9 889.95 593.64 SP333 Ac-LTF$r8EF4cooh6clWAQCba$SANleA- 342 1918.96 961.27 1919.97 960.49 640.66 NH2 SP334 Ac-LTF$r8EF4cooh6clWAQCba$SANleA- 343 iso2 1918.96 961.27 1919.97 960.49 640.66 NH2 SP335 Ac-LTF$r8EF4cooh6clWAQCba$AAIa- 344 1902.97 953.03 1903.98 952.49 635.33 NH2 SP336 Ac-LTF$r8EF4cooh6clWAQCba$AAIa- 345 iso2 1902.97 953.13 1903.98 952.49 635.33 NH2 SP337 Ac-LTF$r8AY6clWAQL$AAAAAa-NH2 346 1905 954.61 1906.01 953.51 636.01 SP338 Ac-LTF$r8AY6clWAQL$AAAAAa-NH2 347 iso2 1905 954.9 1906.01 953.51 636.01 SP339 Ac-F$r8AY6clWEAL$AAAAAAa-NH2 348 1762.89 883.01 1763.9 882.45 588.64 SP340 Ac-ETF$r8EYWAQL$AAAAAa-NH2 349 1945 974.31 1946.01 973.51 649.34 SP341 Ac-ETF$r8EYWAQL$AAAAAa-NH2 350 iso2 1945 974.49 1946.01 973.51 649.34 SP342 Ac-LTF$r8EYWAQL$AAAAAAa-NH2 351 2000.08 1001.6 2001.09 1001.05 667.7 SP343 Ac-LTF$r8EYWAQL$AAAAAAa-NH2 352 iso2 2000.08 1001.6 2001.09 1001.05 667.7 SP344 Ac-LTF$r8AYWAQL$AANleAAa-NH2 353 1913.08 958.58 1914.09 957.55 638.7 SP345 Ac-LTF$r8AYWAQL$AANleAAa-NH2 354 iso2 1913.08 958.58 1914.09 957.55 638.7 SP346 Ac-LTF$r8EYWAQCba$AAAAAa-NH2 355 1941.04 972.55 1942.05 971.53 648.02 SP347 Ac-LTF$r8EYWAQCba$AAAAAa-NH2 356 iso2 1941.04 972.55 1942.05 971.53 648.02 SP348 Ac-LTF$r8EF4coohWAQCba$AAAAAa- 357 1969.04 986.33 1970.05 985.53 657.35 NH2 SP349 Ac-LTF$r8EF4coohWAQCba$AAAAAa- 358 iso2 1969.04 986.06 1970.05 985.53 657.35 NH2 SP350 Ac-LTF$r8EYWSQCba$AAAAAa-NH2 359 1957.04 980.04 1958.05 979.53 653.35 SP351 Ac-LTF$r8EYWSQCba$AAAAAa-NH2 360 iso2 1957.04 980.04 1958.05 979.53 653.35 SP352 Ac-LTF$r8EYWAQCba$SAAa-NH2 361 1814.96 909 1815.97 908.49 605.99 SP353 Ac-LTF$r8EYWAQCba$SAAa-NH2 362 iso2 1814.96 909 1815.97 908.49 605.99 SP354 Ac-ALTF$r8EYWAQCba$SAAa-NH2 363 1886 944.52 1887.01 944.01 629.67 SP355 Ac-ALTF$r8EYWAQCba$SAAa-NH2 364 iso2 1886 944.98 1887.01 944.01 629.67 SP356 Ac-ALTF$r8EYWAQCba$SAAAa-NH2 365 1957.04 980.04 1958.05 979.53 653.35 SP357 Ac-ALTF$r8EYWAQCba$SAAAa-NH2 366 iso2 1957.04 980.04 1958.05 979.53 653.35 SP358 Ac-AALTF$r8EYWAQCba$SAAAa-NH2 367 2028.07 1016.1 2029.08 1015.04 677.03 SP359 Ac-AALTF$r8EYWAQCba$SAAAa-NH2 368 iso2 2028.07 1015.57 2029.08 1015.04 677.03 SP360 Ac-RTF$r8EYWAQCba$SAA-NH2 369 1786.94 895.03 1787.95 894.48 596.65 SP361 Ac-LRF$r8EYWAQCba$SAA-NH2 370 1798.98 901.51 1799.99 900.5 600.67 SP362 Ac-LTF$r8EYWRQCba$SAA-NH2 371 1828.99 916.4 1830 915.5 610.67 SP363 Ac-LTF$r8EYWARCba$SAA-NH2 372 1771.97 887.63 1772.98 886.99 591.66 SP364 Ac-LTF$r8EYWAQCba$RAA-NH2 373 1812.99 908.08 1814 907.5 605.34 SP365 Ac-LTF$r8EYWAQCba$SRA-NH2 374 1828.99 916.12 1830 915.5 610.67 SP366 Ac-LTF$r8EYWAQCba$SAR-NH2 375 1828.99 916.12 1830 915.5 610.67 SP367 5-FAM-BaLTF$r8EYWAQCba$SAA-NH2 376 2131 1067.09 2132.01 1066.51 711.34 SP368 5-FAM-BaLTF$r8AYWAQL$AANleA- 377 2158.08 1080.6 2159.09 1080.05 720.37 NH2 SP369 Ac-LAF$r8EYWAQL$AANleA-NH2 378 1799 901.05 1800.01 900.51 600.67 SP370 Ac-ATF$r8EYWAQL$AANleA-NH2 379 1786.97 895.03 1787.98 894.49 596.66 SP371 Ac-AAF$r8EYWAQL$AANleA-NH2 380 1756.96 880.05 1757.97 879.49 586.66 SP372 Ac-AAAF$r8EYWAQL$AANleA-NH2 381 1827.99 915.57 1829 915 610.34 SP373 Ac-AAAAF$r8EYWAQL$AANleA-NH2 382 1899.03 951.09 1900.04 950.52 634.02 SP374 Ac-AATF$r8EYWAQL$AANleA-NH2 383 1858 930.92 1859.01 930.01 620.34 SP375 Ac-AALTF$r8EYWAQL$AANleA-NH2 384 1971.09 987.17 1972.1 986.55 658.04 SP376 Ac-AAALTF$r8EYWAQL$AANleA-NH2 385 2042.12 1023.15 2043.13 1022.07 681.71 SP377 Ac-LTF$r8EYWAQL$AANleAA-NH2 386 1900.05 952.02 1901.06 951.03 634.36 SP378 Ac-ALTF$r8EYWAQL$AANleAA-NH2 387 1971.09 987.63 1972.1 986.55 658.04 SP379 Ac-AALTF$r8EYWAQL$AANleAA-NH2 388 2042.12 1022.69 2043.13 1022.07 681.71 SP380 Ac-LTF$r8EYWAQCba$AANleAA-NH2 389 1912.05 958.03 1913.06 957.03 638.36 SP381 Ac-LTF$r8EYWAQhL$AANleAA-NH2 390 1914.07 958.68 1915.08 958.04 639.03 SP382 Ac-ALTF$r8EYWAQhL$AANleAA-NH2 391 1985.1 994.1 1986.11 993.56 662.71 SP383 Ac-LTF$r8ANmYWAQL$AANleA-NH2 392 1785.02 894.11 1786.03 893.52 596.01 SP384 Ac-LTF$r8ANmYWAQL$AANleA-NH2 393 iso2 1785.02 894.11 1786.03 893.52 596.01 SP385 Ac-LTF$r8AYNmWAQL$AANleA-NH2 394 1785.02 894.11 1786.03 893.52 596.01 SP386 Ac-LTF$r8AYNmWAQL$AANleA-NH2 395 iso2 1785.02 894.11 1786.03 893.52 596.01 SP387 Ac-LTF$r8AYAmwAQL$AANleA-NH2 396 1785.02 894.01 1786.03 893.52 596.01 SP388 Ac-LTF$r8AYAmwAQL$AANleA-NH2 397 iso2 1785.02 894.01 1786.03 893.52 596.01 SP389 Ac-LTF$r8AYWAibQL$AANleA-NH2 398 1785.02 894.01 1786.03 893.52 596.01 SP390 Ac-LTF$r8AYWAibQL$AANleA-NH2 399 iso2 1785.02 894.01 1786.03 893.52 596.01 SP391 Ac-LTF$r8AYWAQL$AAibNleA-NH2 400 1785.02 894.38 1786.03 893.52 596.01 SP392 Ac-LTF$r8AYWAQL$AAibNleA-NH2 401 iso2 1785.02 894.38 1786.03 893.52 596.01 SP393 Ac-LTF$r8AYWAQL$AaNleA-NH2 402 1771.01 887.54 1772.02 886.51 591.34 SP394 Ac-LTF$r8AYWAQL$AaNleA-NH2 403 iso2 1771.01 887.54 1772.02 886.51 591.34 SP395 Ac-LTF$r8AYWAQL$ASarNleA-NH2 404 1771.01 887.35 1772.02 886.51 591.34 SP396 Ac-LTF$r8AYWAQL$ASarNleA-NH2 405 iso2 1771.01 887.35 1772.02 886.51 591.34 SP397 Ac-LTF$r8AYWAQL$AANleAib-NH2 406 1785.02 894.75 1786.03 893.52 596.01 SP398 Ac-LTF$r8AYWAQL$AANleAib-NH2 407 iso2 1785.02 894.75 1786.03 893.52 596.01 SP399 Ac-LTF$r8AYWAQL$AANleNmA-NH2 408 1785.02 894.6 1786.03 893.52 596.01 SP400 Ac-LTF$r8AYWAQL$AANleNmA-NH2 409 iso2 1785.02 894.6 1786.03 893.52 596.01 SP401 Ac-LTF$r8AYWAQL$AANleSar-NH2 410 1771.01 886.98 1772.02 886.51 591.34 SP402 Ac-LTF$r8AYWAQL$AANleSar-NH2 411 iso2 1771.01 886.98 1772.02 886.51 591.34 SP403 Ac-LTF$r8AYWAQL$AANleAAib-NH2 412 1856.06 1857.07 929.04 619.69 SP404 Ac-LTF$r8AYWAQL$AANleAAib-NH2 413 iso2 1856.06 1857.07 929.04 619.69 SP405 Ac-LTF$r8AYWAQL$AANleANmA-NH2 414 1856.06 930.37 1857.07 929.04 619.69 SP406 Ac-LTF$r8AYWAQL$AANleANmA-NH2 415 iso2 1856.06 930.37 1857.07 929.04 619.69 SP407 Ac-LTF$r8AYWAQL$AANleAa-NH2 416 1842.04 922.69 1843.05 922.03 615.02 SP408 Ac-LTF$r8AYWAQL$AANleAa-NH2 417 iso2 1842.04 922.69 1843.05 922.03 615.02 SP409 Ac-LTF$r8AYWAQL$AANleASar-NH2 418 1842.04 922.6 1843.05 922.03 615.02 SP410 Ac-LTF$r8AYWAQL$AANleASar-NH2 419 iso2 1842.04 922.6 1843.05 922.03 615.02 SP411 Ac-LTF$/r8AYWAQL$/AANleA-NH2 420 1799.04 901.14 1800.05 900.53 600.69 SP412 Ac-LTFAibAYWAQLAibAANleA-NH2 421 1648.9 826.02 1649.91 825.46 550.64 SP413 Ac-LTF$r8Cou4YWAQL$AANleA-NH2 422 1975.05 989.11 1976.06 988.53 659.36 SP414 Ac-LTF$r8Cou4YWAQL$AANleA-NH2 423 iso2 1975.05 989.11 1976.06 988.53 659.36 SP415 Ac-LTF$r8AYWCou4QL$AANleA-NH2 424 1975.05 989.11 1976.06 988.53 659.36 SP416 Ac-LTF$r8AYWAQL$Cou4ANleA-NH2 425 1975.05 989.57 1976.06 988.53 659.36 SP417 Ac-LTF$r8AYWAQL$Cou4ANleA-NH2 426 iso2 1975.05 989.57 1976.06 988.53 659.36 SP418 Ac-LTF$r8AYWAQL$ACou4NleA-NH2 427 1975.05 989.57 1976.06 988.53 659.36 SP419 Ac-LTF$r8AYWAQL$ACou4NleA-NH2 428 iso2 1975.05 989.57 1976.06 988.53 659.36 SP420 Ac-LTF$r8AYWAQL$AANleA-OH 429 1771.99 887.63 1773 887 591.67 SP421 Ac-LTF$r8AYWAQL$AANleA-OH 430 iso2 1771.99 887.63 1773 887 591.67 SP422 Ac-LTF$r8AYWAQL$AANleA-NHnPr 431 1813.05 908.08 1814.06 907.53 605.36 SP423 Ac-LTF$r8AYWAQL$AANleA-NHnPr 432 iso2 1813.05 908.08 1814.06 907.53 605.36 SP424 Ac-LTF$r8AYWAQL$AANleA- 433 1855.1 929.17 1856.11 928.56 619.37 NHnBu33Me SP425 Ac-LTF$r8AYWAQL$AANleA- 434 iso2 1855.1 929.17 1856.11 928.56 619.37 NHnBu33Me SP426 Ac-LTF$r8AYWAQL$AANleA-NHHex 435 1855.1 929.17 1856.11 928.56 619.37 SP427 Ac-LTF$r8AYWAQL$AANleA-NHHex 436 iso2 1855.1 929.17 1856.11 928.56 619.37 SP428 Ac-LTA$r8AYWAQL$AANleA-NH2 437 1694.98 849.33 1695.99 848.5 566 SP429 Ac-LThL$r8AYWAQL$AANleA-NH2 438 1751.04 877.09 1752.05 876.53 584.69 SP430 Ac-LTF$r8AYAAQL$AANleA-NH2 439 1655.97 829.54 1656.98 828.99 553 SP431 Ac-LTF$r8AY2NalAQL$AANleA-NH2 440 1782.01 892.63 1783.02 892.01 595.01 SP432 Ac-LTF$r8EYWCou4QCba$SAA-NH2 441 1947.97 975.8 1948.98 974.99 650.33 SP433 Ac-LTF$r8EYWCou7QCba$SAA-NH2 442 16.03 974.9 17.04 9.02 6.35 SP434 Ac-LTF%r8EYWAQCba%SAA-NH2 443 1745.94 874.8 1746.95 873.98 582.99 SP435 Dmaac-LTF$r8EYWAQCba$SAA-NH2 444 1786.97 894.8 1787.98 894.49 596.66 SP436 Dmaac-LTF$r8AYWAQL$AAAAAa-NH2 445 1914.08 958.2 1915.09 958.05 639.03 SP437 Dmaac-LTF$r8AYWAQL$AAAAAa-NH2 446 iso2 1914.08 958.2 1915.09 958.05 639.03 SP438 Dmaac-LTF$r8EYWAQL$AAAAAa-NH2 447 1972.08 987.3 1973.09 987.05 658.37 SP439 Dmaac-LTF$r8EYWAQL$AAAAAa-NH2 448 iso2 1972.08 987.3 1973.09 987.05 658.37 SP440 Dmaac-LTF$r8EF4coohWAQCba$AAIa- 449 1912.05 957.4 1913.06 957.03 638.36 NH2 SP441 Dmaac-LTF$r8EF4coohWAQCba$AAIa- 450 iso2 1912.05 957.4 1913.06 957.03 638.36 NH2 SP442 Dmaac-LTF$r8AYWAQL$AANleA-NH2 451 1814.05 908.3 1815.06 908.03 605.69 SP443 Dmaac-LTF$r8AYWAQL$AANleA-NH2 452 iso2 1814.05 908.3 1815.06 908.03 605.69 SP444 Ac-LTF%r8AYWAQL%AANleA-NH2 453 1773.02 888.37 1774.03 887.52 592.01 SP445 Ac-LTF%r8EYWAQL%AAAAAa-NH2 454 1931.06 966.4 1932.07 966.54 644.69 SP446 Cou6BaLTF$r8EYWAQhL$SAA-NH2 455 2018.05 1009.9 2019.06 1010.03 673.69 SP447 Cou8BaLTF$r8EYWAQhL$SAA-NH2 456 1962.96 982.34 1963.97 982.49 655.32 SP448 Ac-LTF4l$r8EYWAQL$AAAAAa-NH2 457 2054.93 1028.68 2055.94 1028.47 685.98 SP449 Ac-LTF$r8EYWAQL$AAAAAa-NH2 458 1929.04 966.17 1930.05 965.53 644.02 SP550 Ac-LTF$r8EYWAQL$AAAAAa-OH 459 1930.02 966.54 1931.03 966.02 644.35 SP551 Ac-LTF$r8EYWAQL$AAAAAa-OH 460 iso2 1930.02 965.89 1931.03 966.02 644.35 SP552 Ac-LTF$r8EYWAEL$AAAAAa-NH2 461 1930.02 966.82 1931.03 966.02 644.35 SP553 Ac-LTF$r8EYWAEL$AAAAAa-NH2 462 iso2 1930.02 966.91 1931.03 966.02 644.35 SP554 Ac-LTF$r8EYWAEL$AAAAAa-OH 463 1931.01 967.28 1932.02 966.51 644.68 SP555 Ac-LTF$r8EY6clWAQL$AAAAAa-NH2 464 1963 983.28 1964.01 982.51 655.34 SP556 Ac-LTF$r8EF4bOH2WAQL$AAAAAa- 465 1957.05 980.04 1958.06 979.53 653.36 NH2 SP557 Ac-AAALTF$r8EYWAQL$AAAAAa-NH2 466 2142.15 1072.83 2143.16 1072.08 715.06 SP558 Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2 467 1965.02 984.3 1966.03 983.52 656.01 SP559 Ac-RTF$r8EYWAQL$AAAAAa-NH2 468 1972.06 987.81 1973.07 987.04 658.36 SP560 Ac-LTA$r8EYWAQL$AAAAAa-NH2 469 1853.01 928.33 1854.02 927.51 618.68 SP561 Ac-LTF$r8EYWAibQL$AAAAAa-NH2 470 1943.06 973.48 1944.07 972.54 648.69 SP562 Ac-LTF$r8EYWAQL$AAibAAAa-NH2 471 1943.06 973.11 1944.07 972.54 648.69 SP563 Ac-LTF$r8EYWAQL$AAAibAAa-NH2 472 1943.06 973.48 1944.07 972.54 648.69 SP564 Ac-LTF$r8EYWAQL$AAAAibAa-NH2 473 1943.06 973.48 1944.07 972.54 648.69 SP565 Ac-LTF$r8EYWAQL$AAAAAiba-NH2 474 1943.06 973.38 1944.07 972.54 648.69 SP566 Ac-LTF$r8EYWAQL$AAAAAiba-NH2 475 iso2 1943.06 973.38 1944.07 972.54 648.69 SP567 Ac-LTF$r8EYWAQL$AAAAAAib-NH2 476 1943.06 973.01 1944.07 972.54 648.69 SP568 Ac-LTF$r8EYWAQL$AaAAAa-NH2 477 1929.04 966.54 1930.05 965.53 644.02 SP569 Ac-LTF$r8EYWAQL$AAaAAa-NH2 478 1929.04 966.35 1930.05 965.53 644.02 SP570 Ac-LTF$r8EYWAQL$AAAaAa-NH2 479 1929.04 966.54 1930.05 965.53 644.02 SP571 Ac-LTF$r8EYWAQL$AAAaAa-NH2 480 iso2 1929.04 966.35 1930.05 965.53 644.02 SP572 Ac-LTF$r8EYWAQL$AAAAaa-NH2 481 1929.04 966.35 1930.05 965.53 644.02 SP573 Ac-LTF$r8EYWAQL$AAAAAA-NH2 482 1929.04 966.35 1930.05 965.53 644.02 SP574 Ac-LTF$r8EYWAQL$ASarAAAa-NH2 483 1929.04 966.54 1930.05 965.53 644.02 SP575 Ac-LTF$r8EYWAQL$AASarAAa-NH2 484 1929.04 966.35 1930.05 965.53 644.02 SP576 Ac-LTF$r8EYWAQL$AAASarAa-NH2 485 1929.04 966.35 1930.05 965.53 644.02 SP577 Ac-LTF$r8EYWAQL$AAAASara-NH2 486 1929.04 966.35 1930.05 965.53 644.02 SP578 Ac-LTF$r8EYWAQL$AAAAASar-NH2 487 1929.04 966.08 1930.05 965.53 644.02 SP579 Ac-7LTF$r8EYWAQL$AAAAAa-NH2 488 1918.07 951.99 1919.08 960.04 640.37 SP581 Ac-TF$r8EYWAQL$AAAAAa-NH2 489 1815.96 929.85 1816.97 908.99 606.33 SP582 Ac-F$r8EYWAQL$AAAAAa-NH2 490 1714.91 930.92 1715.92 858.46 572.64 SP583 Ac-LVF$r8EYWAQL$AAAAAa-NH2 491 1927.06 895.12 1928.07 964.54 643.36 SP584 Ac-AAF$r8EYWAQL$AAAAAa-NH2 492 1856.98 859.51 1857.99 929.5 620 SP585 Ac-LTF$r8EYWAQL$AAAAa-NH2 493 1858 824.08 1859.01 930.01 620.34 SP586 Ac-LTF$r8EYWAQL$AAAa-NH2 494 1786.97 788.56 1787.98 894.49 596.66 SP587 Ac-LTF$r8EYWAQL$AAa-NH2 495 1715.93 1138.57 1716.94 858.97 572.98 SP588 Ac-LTF$r8EYWAQL$Aa-NH2 496 1644.89 1144.98 1645.9 823.45 549.3 SP589 Ac-LTF$r8EYWAQL$a-NH2 497 1573.85 1113.71 1574.86 787.93 525.62 SP590 Ac-LTF$r8EYWAQL$AAA-OH 498 1716.91 859.55 1717.92 859.46 573.31 SP591 Ac-LTF$r8EYWAQL$A-OH 499 1574.84 975.14 1575.85 788.43 525.95 SP592 Ac-LTF$r8EYWAQL$AAA-NH2 500 1715.93 904.75 1716.94 858.97 572.98 SP593 Ac-LTF$r8EYWAQCba$SAA-OH 501 1744.91 802.49 1745.92 873.46 582.64 SP594 Ac-LTF$r8EYWAQCba$S-OH 502 1602.83 913.53 1603.84 802.42 535.28 SP595 Ac-LTF$r8EYWAQCba$S-NH2 503 1601.85 979.58 1602.86 801.93 534.96 SP596 4-FBzl-LTF$r8EYWAQL$AAAAAa-NH2 504 2009.05 970.52 2010.06 1005.53 670.69 SP597 4-FBzl-LTF$r8EYWAQCba$SAA-NH2 505 1823.93 965.8 1824.94 912.97 608.98 SP598 Ac-LTF$r8RYWAQL$AAAAAa-NH2 506 1956.1 988.28 1957.11 979.06 653.04 SP599 Ac-LTF$r8HYWAQL$AAAAAa-NH2 507 1937.06 1003.54 1938.07 969.54 646.69 SP600 Ac-LTF$r8QYWAQL$AAAAAa-NH2 508 1928.06 993.92 1929.07 965.04 643.69 SP601 Ac-LTF$r8CitYWAQL$AAAAAa-NH2 509 1957.08 987 1958.09 979.55 653.37 SP602 Ac-LTF$r8GlaYWAQL$AAAAAa-NH2 510 1973.03 983 1974.04 987.52 658.68 SP603 Ac-LTF$r8F4gYWAQL$AAAAAa-NH2 511 2004.1 937.86 2005.11 1003.06 669.04 SP604 Ac-LTF$r82mRYWAQL$AAAAAa-NH2 512 1984.13 958.58 1985.14 993.07 662.38 SP605 Ac-LTF$r8ipKYWAQL$AAAAAa-NH2 513 1970.14 944.52 1971.15 986.08 657.72 SP606 Ac-LTF$r8F4NH2YWAQL$AAAAAa- 514 1962.08 946 1963.09 982.05 655.03 NH2 SP607 Ac-LTF$r8EYWAAL$AAAAAa-NH2 515 1872.02 959.32 1873.03 937.02 625.01 SP608 Ac-LTF$r8EYWALL$AAAAAa-NH2 516 1914.07 980.88 1915.08 958.04 639.03 SP609 Ac-LTF$r8EYWAAibL$AAAAAa-NH2 517 1886.03 970.61 1887.04 944.02 629.68 SP610 Ac-LTF$r8EYWASL$AAAAAa-NH2 518 1888.01 980.51 1889.02 945.01 630.34 SP611 Ac-LTF$r8EYWANL$AAAAAa-NH2 519 1915.02 1006.41 1916.03 958.52 639.35 SP612 Ac-LTF$r8EYWACitL$AAAAAa-NH2 520 1958.07 1959.08 980.04 653.7 SP613 Ac-LTF$r8EYWAHL$AAAAAa-NH2 521 1938.04 966.24 1939.05 970.03 647.02 SP614 Ac-LTF$r8EYWARL$AAAAAa-NH2 522 1957.08 1958.09 979.55 653.37 SP615 Ac-LTF$r8EpYWAQL$AAAAAa-NH2 523 2009.01 2010.02 1005.51 670.68 SP616 Cbm-LTF$r8EYWAQCba$SAA-NH2 524 1590.85 1591.86 796.43 531.29 SP617 Cbm-LTF$r8EYWAQL$AAAAAa-NH2 525 1930.04 1931.05 966.03 644.35 SP618 Ac-LTF$r8EYWAQL$SAAAAa-NH2 526 1945.04 1005.11 1946.05 973.53 649.35 SP619 Ac-LTF$r8EYWAQL$AAAASa-NH2 527 1945.04 986.52 1946.05 973.53 649.35 SP620 Ac-LTF$r8EYWAQL$SAAASa-NH2 528 1961.03 993.27 1962.04 981.52 654.68 SP621 Ac-LTF$r8EYWAQTba$AAAAAa-NH2 529 1943.06 983.1 1944.07 972.54 648.69 SP622 Ac-LTF$r8EYWAQAdm$AAAAAa-NH2 530 2007.09 990.31 2008.1 1004.55 670.04 SP623 Ac-LTF$r8EYWAQCha$AAAAAa-NH2 531 1969.07 987.17 1970.08 985.54 657.36 SP624 Ac-LTF$r8EYWAQhCha$AAAAAa-NH2 532 1983.09 1026.11 1984.1 992.55 662.04 SP625 Ac-LTF$r8EYWAQF$AAAAAa-NH2 533 1963.02 957.01 1964.03 982.52 655.35 SP626 Ac-LTF$r8EYWAQhF$AAAAAa-NH2 534 1977.04 1087.81 1978.05 989.53 660.02 SP627 Ac-LTF$r8EYWAQL$AANleAAa-NH2 535 1971.09 933.45 1972.1 986.55 658.04 SP628 Ac-LTF$r8EYWAQAdm$AANleAAa-NH2 536 2049.13 1017.97 2050.14 1025.57 684.05 SP629 4-FBz-BaLTF$r8EYWAQL$AAAAAa- 537 2080.08 2081.09 1041.05 694.37 NH2 SP630 4-FBz-BaLTF$r8EYWAQCba$SAA-NH2 538 1894.97 1895.98 948.49 632.66 SP631 Ac-LTF$r5EYWAQL$s8AAAAAa-NH2 539 1929.04 1072.68 1930.05 965.53 644.02 SP632 Ac-LTF$r5EYWAQCba$s8SAA-NH2 540 1743.92 1107.79 1744.93 872.97 582.31 SP633 Ac-LTF$r8EYWAQL$AAhhLAAa-NH2 541 1999.12 2000.13 1000.57 667.38 SP634 Ac-LTF$r8EYWAQL$AAAAAAAa-NH2 542 2071.11 2072.12 1036.56 691.38 SP635 Ac-LTF$r8EYWAQL$AAAAAAAAa-NH2 543 2142.15 778.1 2143.16 1072.08 715.06 SP636 Ac-LTF$r8EYWAQL$AAAAAAAAAa- 544 2213.19 870.53 2214.2 1107.6 738.74 NH2 SP637 Ac-LTA$r8EYAAQCba$SAA-NH2 545 1552.85 1553.86 777.43 518.62 SP638 Ac-LTA$r8EYAAQL$AAAAAa-NH2 546 1737.97 779.45 1738.98 869.99 580.33 SP639 Ac-LTF$r8EPmpWAQL$AAAAAa-NH2 547 2007.03 779.54 2008.04 1004.52 670.02 SP640 Ac-LTF$r8EPmpWAQCba$SAA-NH2 548 1821.91 838.04 1822.92 911.96 608.31 SP641 Ac-ATF$r8HYWAQL$S-NH2 549 1555.82 867.83 1556.83 778.92 519.61 SP642 Ac-LTF$r8HAWAQL$S-NH2 550 1505.84 877.91 1506.85 753.93 502.95 SP643 Ac-LTF$r8HYWAQA$S-NH2 551 1555.82 852.52 1556.83 778.92 519.61 SP644 Ac-LTF$r8EYWAQCba$SA-NH2 552 1672.89 887.18 1673.9 837.45 558.64 SP645 Ac-LTF$r8EYWAQL$SAA-NH2 553 1731.92 873.32 1732.93 866.97 578.31 SP646 Ac-LTF$r8HYWAQCba$SAA-NH2 554 1751.94 873.05 1752.95 876.98 584.99 SP647 Ac-LTF$r8SYWAQCba$SAA-NH2 555 1701.91 844.88 1702.92 851.96 568.31 SP648 Ac-LTF$r8RYWAQCba$SAA-NH2 556 1770.98 865.58 1771.99 886.5 591.33 SP649 Ac-LTF$r8KYWAQCba$SAA-NH2 557 1742.98 936.57 1743.99 872.5 582 SP650 Ac-LTF$r8QYWAQCba$SAA-NH2 558 1742.94 930.93 1743.95 872.48 581.99 SP651 Ac-LTF$r8EYWAACba$SAA-NH2 559 1686.9 1032.45 1687.91 844.46 563.31 SP652 Ac-LTF$r8EYWAQCba$AAA-NH2 560 1727.93 895.46 1728.94 864.97 576.98 SP653 Ac-LTF$r8EYWAQL$AAAAA-OH 561 1858.99 824.54 1860 930.5 620.67 SP654 Ac-LTF$r8EYWAQL$AAAA-OH 562 1787.95 894.48 1788.96 894.98 596.99 SP655 Ac-LTF$r8EYWAQL$AA-OH 563 1645.88 856 1646.89 823.95 549.63 SP656 Ac-LTF$r8AF4bOH2WAQL$AAAAAa- 564 NH2 SP657 Ac-LTF$r8AF4bOH2WAAL$AAAAAa- 565 NH2 SP658 Ac-LTF$r8EF4bOH2WAQCba$SAA-NH2 566 SP659 Ac-LTF$r8ApYWAQL$AAAAAa-NH2 567 SP660 Ac-LTF$r8ApYWAAL$AAAAAa-NH2 568 SP661 Ac-LTF$r8EpYWAQCba$SAA-NH2 569 SP662 Ac-LTF$rda6AYWAQL$da5AAAAAa- 570 1974.06 934.44 NH2 SP663 Ac-LTF$rda6EYWAQCba$da5SAA-NH2 571 1846.95 870.52 869.94 SP664 Ac-LTF$rda6EYWAQL$da5AAAAAa- 572 NH2 SP665 Ac-LTF$ra9EYWAQL$a6AAAAAa-NH2 573 936.57 935.51 SP666 Ac-LTF$ra9EYWAQL$a6AAAAAa-NH2 574 SP667 Ac-LTF$ra9EYWAQCba$a6SAA-NH2 575 SP668 Ac-LTA$ra9EYWAQCba$a6SAA-NH2 576 SP669 5-FAM-BaLTF$ra9EYWAQCba$a6SAA- 577 NH2 SP670 5-FAM-BaLTF$r8EYWAQL$AAAAAa- 578 2316.11 NH2 SP671 5-FAM-BaLTF$/r8EYWAQL$/AAAAAa- 579 2344.15 NH2 SP672 5-FAM-BaLTA$r8EYWAQL$AAAAAa- 580 2240.08 NH2 SP673 5-FAM-BaLTF$r8AYWAQL$AAAAAa- 581 2258.11 NH2 SP674 5-FAM-BaATF$r8EYWAQL$AAAAAa- 582 2274.07 NH2 SP675 5-FAM-BaLAF$r8EYWAQL$AAAAAa- 583 2286.1 NH2 SP676 5-FAM-BaLTF$r8EAWAQL$AAAAAa- 584 2224.09 NH2 SP677 5-FAM-BaLTF$r8EYAAQL$AAAAAa- 585 2201.07 NH2 SP678 5-FAM-BaLTA$r8EYAAQL$AAAAAa- 586 2125.04 NH2 SP679 5-FAM-BaLTF$r8EYWAAL$AAAAAa- 587 2259.09 NH2 SP680 5-FAM-BaLTF$r8EYWAQA$AAAAAa- 588 2274.07 NH2 SP681 5-FAM-BaLTF$/r8EYWAQCba$/SAA- 589 2159.03 NH2 SP682 5-FAM-BaLTA$r8EYWAQCba$SAA-NH2 590 2054.97 SP683 5-FAM-BaLTF$r8EYAAQCba$SAA-NH2 591 2015.96 SP684 5-FAM-BaLTA$r8EYAAQCba$SAA-NH2 592 1939.92 SP685 5-FAM-BaQSQQTF$r8NLWRLL$QN-NH2 593 2495.23 SP686 5-TAMRA-BaLTF$r8EYWAQCba$SAA- 594 2186.1 NH2 SP687 5-TAMRA-BaLTA$r8EYWAQCba$SAA- 595 2110.07 NH2 SP688 5-TAMRA-BaLTF$r8EYAAQCba$SAA- 596 2071.06 NH2 SP689 5-TAMRA-BaLTA$r8EYAAQCba$SAA- 597 1995.03 NH2 SP690 5-TAMRA-BaLTF$/r8EYWAQCba$/SAA- 598 2214.13 NH2 SP691 5-TAMRA- 599 2371.22 BaLTF$r8EYWAQL$AAAAAa-NH2 SP692 5-TAMRA- 600 2295.19 BaLTA$r8EYWAQL$AAAAAa-NH2 SP693 5-TAMRA- 601 2399.25 BaLTF$/r8EYWAQLVAAAAAa-NH2 SP694 Ac-LTF$r8EYWCou7QCba$SAA-OH 602 1947.93 SP695 Ac-LTF$r8EYWCou7QCba$S-OH 603 1805.86 SP696 Ac-LTA$r8EYWCou7QCba$SAA-NH2 604 1870.91 SP697 Ac-LTF$r8EYACou7QCba$SAA-NH2 605 1831.9 SP698 Ac-LTA$r8EYACou7QCba$SAA-NH2 606 1755.87 SP699 Ac-LTF$/r8EYWCou7QCbaS/SAA-NH2 607 1974.98 SP700 Ac-LTF$r8EYWCou7QL$AAAAAa-NH2 608 2132.06 SP701 Ac-LTF$/r8EYWCou7QL$/AAAAAa-NH2 609 2160.09 SP702 Ac-LTF$r8EYWCou7QL$AAAAA-OH 610 2062.01 SP703 Ac-LTF$r8EYWCou7QL$AAAA-OH 611 1990.97 SP704 Ac-LTF$r8EYWCou7QL$AAA-OH 612 1919.94 SP705 Ac-LTF$r8EYWCou7QL$AA-OH 613 1848.9 SP706 Ac-LTF$r8EYWCou7QL$A-OH 614 1777.86 SP707 Ac-LTF$r8EYWAQL$AAAASa-NH2 615 iso2 974.4 973.53 SP708 Ac-LTF$r8AYWAAL$AAAAAa-NH2 616 iso2 1814.01 908.82 1815.02 908.01 605.68 SP709 Biotin-BaLTF$r8EYWAQL$AAAAAa- 617 2184.14 1093.64 2185.15 1093.08 729.05 NH2 SP710 Ac-LTF$r8HAWAQL$S-NH2 618 iso2 1505.84 754.43 1506.85 753.93 502.95 SP711 Ac-LTF$r8EYWAQCba$SA-NH2 619 iso2 1672.89 838.05 1673.9 837.45 558.64 SP712 Ac-LTF$r8HYWAQCba$SAA-NH2 620 iso2 1751.94 877.55 1752.95 876.98 584.99 SP713 Ac-LTF$r8SYWAQCba$SAA-NH2 621 iso2 1701.91 852.48 1702.92 851.96 568.31 SP714 Ac-LTF$r8RYWAQCba$SAA-NH2 622 iso2 1770.98 887.45 1771.99 886.5 591.33 SP715 Ac-LTF$r8KYWAQCba$SAA-NH2 623 iso2 1742.98 872.92 1743.99 872.5 582 SP716 Ac-LTF$r8EYWAQCba$AAA-NH2 624 iso2 1727.93 865.71 1728.94 864.97 576.98 SP717 Ac-LTF$r8EYWAQL$AAAAAaBaC-NH2 625 2103.09 1053.12 2104.1 1052.55 702.04 SP718 Ac-LTF$r8EYWAQL$AAAAAadPeg4C- 626 2279.19 1141.46 2280.2 1140.6 760.74 NH2 SP719 Ac-LTA$r8AYWAAL$AAAAAa-NH2 627 1737.98 870.43 1738.99 870 580.33 SP720 Ac-LTF$r8AYAAAL$AAAAAa-NH2 628 1698.97 851 1699.98 850.49 567.33 SP721 5-FAM-BaLTF$r8AYWAAL$AAAAAa- 629 2201.09 1101.87 2202.1 1101.55 734.7 NH2 SP722 Ac-LTA$r8AYWAQL$AAAAAa-NH2 630 1795 898.92 1796.01 898.51 599.34 SP723 Ac-LTF$r8AYAAQL$AAAAAa-NH2 631 1755.99 879.49 1757 879 586.34 SP724 Ac-LTF$rda6AYWAAL$da5AAAAAa- 632 1807.97 1808.98 904.99 603.66 NH2 SP725 FITC-BaLTF$r8EYWAQL$AAAAAa-NH2 633 2347.1 1174.49 2348.11 1174.56 783.37 SP726 FITC-BaLTF$r8EYWAQCba$SAA-NH2 634 2161.99 1082.35 2163 1082 721.67 SP733 Ac-LTF$r8EYWAQL$EAAAAa-NH2 635 1987.05 995.03 1988.06 994.53 663.36 SP734 Ac-LTF$r8AYWAQL$EAAAAa-NH2 636 1929.04 966.35 1930.05 965.53 644.02 SP735 Ac-LTF$r8EYWAQL$AAAAAaBaKbio- 637 2354.25 1178.47 2355.26 1178.13 785.76 NH2 SP736 Ac-LTF$r8AYWAAL$AAAAAa-NH2 638 1814.01 908.45 1815.02 908.01 605.68 SP737 Ac-LTF$r8AYAAAL$AAAAAa-NH2 639 iso2 1698.97 850.91 1699.98 850.49 567.33 SP738 Ac-LTF$r8AYAAQL$AAAAAa-NH2 640 iso2 1755.99 879.4 1757 879 586.34 SP739 Ac-LTF$r8EYWAQL$EAAAAa-NH2 641 iso2 1987.05 995.21 1988.06 994.53 663.36 SP740 Ac-LTF$r8AYWAQL$EAAAAa-NH2 642 iso2 1929.04 966.08 1930.05 965.53 644.02 SP741 Ac-LTF$r8EYWAQCba$SAAAAa-NH2 643 1957.04 980.04 1958.05 979.53 653.35 SP742 Ac-LTF$r8EYWAQLStAAA$r5AA-NH2 644 2023.12 1012.83 2024.13 1012.57 675.38 SP743 Ac-LTF$r8EYWAQL$A$AAA$A-NH2 645 2108.17 1055.44 2109.18 1055.09 703.73 SP744 Ac-LTF$r8EYWAQL$AA$AAA$A-NH2 646 2179.21 1090.77 2180.22 1090.61 727.41 SP745 Ac-LTF$r8EYWAQL$AAA$AAA$A-NH2 647 2250.25 1126.69 2251.26 1126.13 751.09 SP746 Ac-AAALTF$r8EYWAQL$AAA-OH 648 1930.02 1931.03 966.02 644.35 SP747 Ac-AAALTF$r8EYWAQL$AAA-NH2 649 1929.04 965.85 1930.05 965.53 644.02 SP748 Ac-AAAALTF$r8EYWAQL$AAA-NH2 650 2000.08 1001.4 2001.09 1001.05 667.7 SP749 Ac-AAAAALTF$r8EYWAQL$AAA-NH2 651 2071.11 1037.13 2072.12 1036.56 691.38 SP750 Ac-AAAAAALTF$r8EYWAQL$AAA- 652 2142.15 2143.16 1072.08 715.06 NH2 SP751 Ac-LTF$rda6EYWAQCba$da6SAA-NH2 653 iso2 1751.89 877.36 1752.9 876.95 584.97 SP752 Ac-t$r5wya$r5f4CF3ekllr-NH2 654 844.25 SP753 Ac-tawy$r5nf4CF3e$r5llr-NH2 655 837.03 SP754 Ac-tawya$r5f4CF3ek$r5lr-NH2 656 822.97 SP755 Ac-tawyanf4CF3e$r5llr$r5a-NH2 657 908.35 SP756 Ac-t$s8wyanf4CF3e$r5llr-NH2 658 858.03 SP757 Ac-tawy$s8nf4CF3ekll$r5a-NH2 659 879.86 SP758 Ac-tawya$s8f4CF3ekllr$r5a-NH2 660 936.38 SP759 Ac-tawy$s8naekll$r5a-NH2 661 844.25 SP760 5-FAM-Batawy$s8nf4CF3ekll$r5a-NH2 662 SP761 5-FAM-Batawy$s8naekll$r5a-NH2 663 SP762 Ac-tawy$s8nf4CF3eall$r5a-NH2 664 SP763 Ac-tawy$s8nf4CF3ekll$r5aaaaa-NH2 665 SP764 Ac-tawy$s8nf4CF3eall$r5aaaaa-NH2 666

Table 3a shows a selection of peptidomimetic macrocycles.

TABLE 3a SEQ ID Exact Found Calc Calc Calc SP Sequence NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP244 Ac-LTF$r8EF4coohWAQCba$SANleA- 667 1885 943.59 1886.01 943.51 629.34 NH2 SP331 Ac-LTF$r8EYWAQL$AAAAAa-NH2 668 iso2 1929.04 966.08 1930.05 965.53 644.02 SP555 Ac-LTF$r8EY6clWAQL$AAAAAa-NH2 669 1963 983.28 1964.01 982.51 655.34 SP557 Ac-AAALTF$r8EYWAQL$AAAAAa-NH2 670 2142.15 1072.83 2143.16 1072.08 715.06 SP558 Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2 671 1965.02 984.3 1966.03 983.52 656.01 SP562 Ac-LTF$r8EYWAQL$AAibAAAa-NH2 672 1943.06 973.11 1944.07 972.54 648.69 SP564 Ac-LTF$r8EYWAQL$AAAAibAa-NH2 673 1943.06 973.48 1944.07 972.54 648.69 SP566 Ac-LTF$r8EYWAQL$AAAAAiba-NH2 674 iso2 1943.06 973.38 1944.07 972.54 648.69 SP567 Ac-LTF$r8EYWAQL$AAAAAAib-NH2 675 1943.06 973.01 1944.07 972.54 648.69 SP572 Ac-LTF$r8EYWAQL$AAAAaa-NH2 676 1929.04 966.35 1930.05 965.53 644.02 SP573 Ac-LTF$r8EYWAQL$AAAAAA-NH2 677 1929.04 966.35 1930.05 965.53 644.02 SP578 Ac-LTF$r8EYWAQL$AAAAASar-NH2 678 1929.04 966.08 1930.05 965.53 644.02 SP551 Ac-LTF$r8EYWAQL$AAAAAa-OH 679 iso2 1930.02 965.89 1931.03 966.02 644.35 SP662 Ac-LTF$rda6AYWAQL$da5AAAAAa-NH2 680 1974.06 934.44 933.49 SP367 5-FAM-BaLTF$r8EYWAQCba$SAA-NH2 681 2131 1067.09 2132.01 1066.51 711.34 SP349 Ac-LTF$r8EF4coohWAQCba$AAAAAa- 682 iso2 1969.04 986.06 1970.05 985.53 657.35 NH2 SP347 Ac-LTF$r8EYWAQCba$AAAAAa-NH2 683 iso2 1941.04 972.55 1942.05 971.53 648.02

Table 3b shows a further selection of peptidomimetic macrocycles.

TABLE 3b SEQ ID Exact Found Calc Calc Calc SP Sequence NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP581 Ac-TF$r8EYWAQL$AAAAAa-NH2 684 1815.96 929.85 1816.97 908.99 606.33 SP582 Ac-F$r8EYWAQL$AAAAAa-NH2 685 1714.91 930.92 1715.92 858.46 572.64 SP583 Ac-LVF$r8EYWAQL$AAAAAa-NH2 686 1927.06 895.12 1928.07 964.54 643.36 SP584 Ac-AAF$r8EYWAQL$AAAAAa-NH2 687 1856.98 859.51 1857.99 929.5 620 SP585 Ac-LTF$r8EYWAQL$AAAAa-NH2 688 1858 824.08 1859.01 930.01 620.34 SP586 Ac-LTF$r8EYWAQL$AAAa-NH2 689 1786.97 788.56 1787.98 894.49 596.66 SP587 Ac-LTF$r8EYWAQL$AAa-NH2 690 1715.93 1138.57 1716.94 858.97 572.98 SP588 Ac-LTF$r8EYWAQL$Aa-NH2 691 1644.89 1144.98 1645.9 823.45 549.3 SP589 Ac-LTF$r8EYWAQL$a-NH2 692 1573.85 1113.71 1574.86 787.93 525.62

In the sequences shown above and elsewhere, the following abbreviations are used: “Nle” represents norleucine, “Aib” represents 2-aminoisobutyric acid, “Ac” represents acetyl, and “Pr” represents propionyl. Amino acids represented as “$” are alpha-Me S5-pentenyl-alanine olefin amino acids connected by an all-carbon crosslinker comprising one double bond. Amino acids represented as “$r5” are alpha-Me R5-pentenyl-alanine olefin amino acids connected by an all-carbon comprising one double bond. Amino acids represented as “$s8” are alpha-Me S8-octenyl-alanine olefin amino acids connected by an all-carbon crosslinker comprising one double bond. Amino acids represented as “$r8” are alpha-Me R8-octenyl-alanine olefin amino acids connected by an all-carbon crosslinker comprising one double bond. “Ahx” represents an aminocyclohexyl linker. The crosslinkers are linear all-carbon crosslinker comprising eight or eleven carbon atoms between the alpha carbons of each amino acid. Amino acids represented as “$/” are alpha-Me S5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/r5” are alpha-Me R5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/s8” are alpha-Me S8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/r8” are alpha-Me R8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “Amw” are alpha-Me tryptophan amino acids. Amino acids represented as “Aml” are alpha-Me leucine amino acids. Amino acids represented as “Amf” are alpha-Me phenylalanine amino acids. Amino acids represented as “2ff” are 2-fluoro-phenylalanine amino acids. Amino acids represented as “3ff” are 3-fluoro-phenylalanine amino acids. Amino acids represented as “St” are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated. Amino acids represented as “St//” are amino acids comprising two pentenyl-alanine olefin side chains that are not crosslinked. Amino acids represented as “% St” are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated via fully saturated hydrocarbon crosslinks. Amino acids represented as “Ba” are beta-alanine. The lower-case character “e” or “z” within the designation of a crosslinked amino acid (e.g. “$er8” or “$zr8”) represents the configuration of the double bond (E or Z, respectively). In other contexts, lower-case letters such as “a” or “f” represent D amino acids (e.g. D-alanine, or D-phenylalanine, respectively). Amino acids designated as “NmW” represent N-methyltryptophan. Amino acids designated as “NmY” represent N-methyltyrosine. Amino acids designated as “NmA” represent N-methylalanine. “Kbio” represents a biotin group attached to the side chain amino group of a lysine residue. Amino acids designated as “Sar” represent sarcosine. Amino acids designated as “Cha” represent cyclohexyl alanine. Amino acids designated as “Cpg” represent cyclopentyl glycine. Amino acids designated as “Chg” represent cyclohexyl glycine. Amino acids designated as “Cba” represent cyclobutyl alanine. Amino acids designated as “F4I” represent 4-iodo phenylalanine. “7L” represents N15 isotopic leucine. Amino acids designated as “F3C1” represent 3-chloro phenylalanine. Amino acids designated as “F4cooh” represent 4-carboxy phenylalanine. Amino acids designated as “F34F2” represent 3,4-difluoro phenylalanine. Amino acids designated as “6clW” represent 6-chloro tryptophan. Amino acids designated as “$rda6” represent alpha-Me R₆-hexynyl-alanine alkynyl amino acids, crosslinked via a dialkyne bond to a second alkynyl amino acid. Amino acids designated as “$da5” represent alpha-Me S5-pentynyl-alanine alkynyl amino acids, wherein the alkyne forms one half of a dialkyne bond with a second alkynyl amino acid. Amino acids designated as “$ra9” represent alpha-Me R₉-nonynyl-alanine alkynyl amino acids, crosslinked via an alkyne metathesis reaction with a second alkynyl amino acid. Amino acids designated as “$a6” represent alpha-Me S6-hexynyl-alanine alkynyl amino acids, crosslinked via an alkyne metathesis reaction with a second alkynyl amino acid. The designation “iso1” or “iso2” indicates that the peptidomimetic macrocycle is a single isomer.

Amino acids designated as “Cit” represent citrulline. Amino acids designated as “Cou4”, “Cou6”, “Cou7” and “Cou8”, respectively, represent the following structures:

In some embodiments, a peptidomimetic macrocycle is obtained in more than one isomer, for example due to the configuration of a double bond within the structure of the crosslinker (E vs Z). In some embodiments, such isomers can or cannot be separated by conventional chromatographic methods. In some embodiments, one isomer has improved biological properties relative to the other isomer. In one embodiment, an E crosslinker olefin isomer of a peptidomimetic macrocycle has better solubility, better target affinity, better in vivo or in vitro efficacy, higher helicity, or improved cell permeability relative to its Z counterpart. In another embodiment, a Z crosslinker olefin isomer of a peptidomimetic macrocycle has better solubility, better target affinity, better in vivo or in vitro efficacy, higher helicity, or improved cell permeability relative to its E counterpart.

Table 3c shows example peptidomimetic macrocycles:

TABLE 3c Structure SEQ ID NO:

163

124

123

108

397

340

454

360

80

78

16

169

324

258

446

358

464

466

467

376

471

473

475

476

481

482

487

572

572

1500

In some embodiments, peptidomimetic macrocycles exclude one or more of the peptidomimetic macrocycles shown in Table 4a:

TABLE 4a Number Sequence SEQ ID NO: 1 L$r5QETFSD$s8WKLLPEN 693 2 LSQ$r5TFSDLW$s8LLPEN 694 3 LSQE$r5FSDLWK$s8LPEN 695 4 LSQET$r5SDLWKL$s8PEN 696 5 LSQETF$r5DLWKLL$s8EN 697 6 LXQETFS$r5LWKLLP$s8N 698 7 LSQETFSD$r5WKLLPE$s8 699 8 LSQQTF$r5DLWKLL$s8EN 700 9 LSQETF$r5DLWKLL$s8QN 701 10 LSQQTF$r5DLWKLL$s8QN 702 11 LSQETF$r5NLWKLL$s8QN 703 12 LSQQTF$r5NLWKLL$s8QN 704 13 LSQQTF$r5NLWRLL$s8QN 705 14 QSQQTF$r5NLWKLL$s8QN 706 15 QSQQTF$r5NLWRLL$s8QN 707 16 QSQQTA$r5NLWRLL$s8QN 708 17 L$r8QETFSD$WKLLPEN 709 18 LSQ$r8TFSDLW$LLPEN 710 19 LSQE$r8FSDLWK$LPEN 711 20 LSQET$r8SDLWKL$PEN 712 21 LSQETF$r8DLWKLL$EN 713 22 LXQETFS$r8LWKLLP$N 714 23 LSQETFSD$r8WKLLPE$ 715 24 LSQQTF$r8DLWKLL$EN 716 25 LSQETF$r8DLWKLL$QN 717 26 LSQQTF$r8DLWKLL$QN 718 27 LSQETF$r8NLWKLL$QN 719 28 LSQQTF$r8NLWKLL$QN 720 29 LSQQTF$r8NLWRLL$QN 721 30 QSQQTF$r8NLWKLL$QN 722 31 QSQQTF$r8NLWRLL$QN 723 32 QSQQTA$r8NLWRLL$QN 724 33 QSQQTF$r8NLWRKK$QN 725 34 QQTF$r8DLWRLL$EN 726 35 QQTF$r8DLWRLL$ 727 36 LSQQTF$DLW$LL 728 37 QQTF$DLW$LL 729 38 QQTA$r8DLWRLL$EN 730 39 QSQQTF$r5NLWRLL$s8QN 731 (dihydroxylated alkylene crosslink) 40 QSQQTA$r5NLWRLL$s8QN 732 (dihydroxylated alkylene crosslink) 41 QSQQTF$r8DLWRLL$QN 733 42 QTF$r8NLWRLL$ 734 43 QSQQTF$NLW$LLPQN 735 44 QS$QTF$NLWRLLPQN 736 45 $TFS$LWKLL 737 46 ETF$DLW$LL 738 47 QTF$NLW$LL 739 48 $SQE$FSNLWKLL 740

-   -   In Table 4a, X represents S or any amino acid. Peptides shown         can comprise an N-terminal capping group such as acetyl or an         additional linker such as beta-alanine between the capping group         and the start of the peptide sequence.

In some embodiments, peptidomimetic macrocycles do not comprise a peptidomimetic macrocycle structure as shown in Table 4a.

In other embodiments, peptidomimetic macrocycles exclude one or more of the peptidomimetic macrocycles shown in Table 4b:

TABLE 4b SEQ ID Exact Observed Number Sequence NO: Mass M + 2 mass (m/e) 1 Ac-LSQETF$r8DLWKLL$EN-NH2 741 2068.13 1035.07 1035.36 2 Ac-LSQETF$r8NLWKLL$QN-NH2 742 2066.16 1034.08 1034.31 3 Ac-LSQQTF$r8NLWRLL$QN-NH2 743 2093.18 1047.59 1047.73 4 Ac-QSQQTF$r8NLWKLL$QN-NH2 744 2080.15 1041.08 1041.31 5 Ac-QSQQTF$r8NLWRLL$QN-NH2 745 2108.15 1055.08 1055.32 6 Ac-QSQQTA$r8NLWRLL$QN-NH2 746 2032.12 1017.06 1017.24 7 Ac-QAibQQTF$r8NLWRLL$QN-NH2 747 2106.17 1054.09 1054.34 8 Ac-QSQQTFSNLWRLLPQN-NH2 748 2000.02 1001.01 1001.26 9 Ac-QSQQTF$/r8NLWRLL$/QN-NH2 749 2136.18 1069.09 1069.37 10 Ac-QSQAibTF$r8NLWRLL$QN-NH2 750 2065.15 1033.58 1033.71 11 Ac-QSQQTF$r8NLWRWAN-NH2 751 2051.13 1026.57 1026.70 12 Ac-ASQQTF$r8NLWRLL$QN-NH2 752 2051.13 1026.57 1026.90 13 Ac-QSQQTF$r8ALWRLL$QN-NH2 753 2065.15 1033.58 1033.41 14 Ac-QSQETF$r8NLWRLL$QN-NH2 754 2109.14 1055.57 1055.70 15 Ac-RSQQTF$r8NLWRLL$QN-NH2 755 2136.20 1069.10 1069.17 16 Ac-RSQQTF$r8NLWRWEN-NH2 756 2137.18 1069.59 1069.75 17 Ac-LSQETFSDLWKLLPEN-NH2 757 1959.99 981.00 981.24 18 Ac-QSQ$TFS$LWRLLPQN-NH2 758 2008.09 1005.05 1004.97 19 Ac-QSQQ$FSN$WRLLPQN-NH2 759 2036.06 1019.03 1018.86 20 Ac-QSQQT$SNL$RLLPQN-NH2 760 1917.04 959.52 959.32 21 Ac-QSQQTF$NLW$LLPQN-NH2 761 2007.06 1004.53 1004.97 22 Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2 762 2310.26 1156.13 1156.52 23 Ac-QSQQTF$r8NLWRLL$RN-NH2 763 2136.20 1069.10 1068.94 24 Ac-QSQRTF$r8NLWRLL$QN-NH2 764 2136.20 1069.10 1068.94 25 Ac-QSQQTF$r8NNleWRLL$QN-NH2 765 2108.15 1055.08 1055.44 26 Ac-QSQQTF$r8NLWRNleL$QN-NH2 766 2108.15 1055.08 1055.84 27 Ac-QSQQTF$r8NLWRLNle$QN-NH2 767 2108.15 1055.08 1055.12 28 Ac-QSQQTY$r8NLWRLL$QN-NH2 768 2124.15 1063.08 1062.92 29 Ac-RAibQQTF$r8NLWRLL$QN-NH2 769 2134.22 1068.11 1068.65 30 Ac-MPRFMDYWEGLN-NH2 770 1598.70 800.35 800.45 31 Ac-RSQQRF$r8NLWRLL$QN-NH2 771 2191.25 1096.63 1096.83 32 Ac-QSQQRF$r8NLWRLL$QN-NH2 772 2163.21 1082.61 1082.87 33 Ac-RAibQQRF$r8NLWRLL$QN-NH2 773 2189.27 1095.64 1096.37 34 Ac-RSQQRF$r8NFWRLL$QN-NH2 774 2225.23 1113.62 1114.37 35 Ac-RSQQRF$r8NYWRLL$QN-NH2 775 2241.23 1121.62 1122.37 36 Ac-RSQQTF$r8NLWQLL$QN-NH2 776 2108.15 1055.08 1055.29 37 Ac-QSQQTF$r8NLWQAmlL$QN-NH2 777 2094.13 1048.07 1048.32 38 Ac-QSQQTF$r8NAmlWRLL$QN-NH2 778 2122.17 1062.09 1062.35 39 Ac-NlePRF$r8DYWEGL$QN-NH2 779 1869.98 935.99 936.20 40 Ac-NlePRF$r8NYWRLL$QN-NH2 780 1952.12 977.06 977.35 41 Ac-RF$r8NLWRLL$Q-NH2 781 1577.96 789.98 790.18 42 Ac-QSQQTF$r8N2ffWRLL$QN-NH2 782 2160.13 1081.07 1081.40 43 Ac-QSQQTF$r8N3ffWRLL$QN-NH2 783 2160.13 1081.07 1081.34 44 Ac-QSQQTF#r8NLWRLL#QN-NH2 784 2080.12 1041.06 1041.34 45 Ac-RSQQTA$r8NLWRLL$QN-NH2 785 2060.16 1031.08 1031.38 46 Ac-QSQQTF%r8NLWRLL%QN-NH2 786 2110.17 1056.09 1056.55 47 HepQSQ$TFSNLWRLLPQN-NH2 787 2051.10 1026.55 1026.82 48 HepQSQ$TF$r8NLWRLL$QN-NH2 788 2159.23 1080.62 1080.89 49 Ac-QSQQTF$r8NL6clWRLL$QN-NH2 789 2142.11 1072.06 1072.35 50 Ac-QSQQTF$r8NLMe6clwRLL$QN-NH2 790 2156.13 1079.07 1079.27 51 Ac-LTFEHYWAQLTS-NH2 791 1535.74 768.87 768.91 52 Ac-LTF$HYW$QLTS-NH2 792 1585.83 793.92 794.17 53 Ac-LTFE$YWASLTS-NH2 793 1520.79 761.40 761.67 54 Ac-LTF$zr8HYWAQL$zS-NH2 794 1597.87 799.94 800.06 55 Ac-LTF$r8HYWRQL$S-NH2 795 1682.93 842.47 842.72 56 Ac-QS$QTFStNLWRLL$s8QN-NH2 796 2145.21 1073.61 1073.90 57 Ac-QSQQTASNLWRLLPQN-NH2 797 1923.99 963.00 963.26 58 Ac-QSQQTA$/r8NLWRLL$/QN-NH2 798 2060.15 1031.08 1031.24 59 Ac-ASQQTF$/r8NLWRLL$/QN-NH2 799 2079.16 1040.58 1040.89 60 Ac-$SQQ$FSNLWRLLAibQN-NH2 800 2009.09 1005.55 1005.86 61 Ac-QS$QTF$NLWRLLAibQN-NH2 801 2023.10 1012.55 1012.79 62 Ac-QSQQ$FSN$WRLLAibQN-NH2 802 2024.06 1013.03 1013.31 63 Ac-QSQQTF$NLW$LLAibQN-NH2 803 1995.06 998.53 998.87 64 Ac-QSQQTFS$LWR$LAibQN-NH2 804 2011.06 1006.53 1006.83 65 Ac-QSQQTFSNLW$LLA$N-NH2 805 1940.02 971.01 971.29 66 Ac-$/SQQ$/FSNLWRLLAibQN-NH2 806 2037.12 1019.56 1019.78 67 Ac-QS$/QTF$/NLWRLLAibQN-NH2 807 2051.13 1026.57 1026.90 68 Ac-QSQQ$/FSN$/WRLLAibQN-NH2 808 2052.09 1027.05 1027.36 69 Ac-QSQQTF$/NLW$/LLAibQN-NH2 809 2023.09 1012.55 1013.82 70 Ac-QSQ$TFS$LWRLLAibQN-NH2 810 1996.09 999.05 999.39 71 Ac-QSQ$/TFS$/LWRLLAibQN-NH2 811 2024.12 1013.06 1013.37 72 Ac-QS$/QTFSt//NLWRLL$/s8QN-NH2 812 2201.27 1101.64 1102.00 73 Ac-$r8SQQTFS$LWRLLAibQN-NH2 813 2038.14 1020.07 1020.23 74 Ac-QSQ$r8TFSNLW$LLAibQN-NH2 814 1996.08 999.04 999.32 75 Ac-QSQQTFS$r8LWRLLA$N-NH2 815 2024.12 1013.06 1013.37 76 Ac-QS$r5QTFStNLW$LLAibQN-NH2 816 2032.12 1017.06 1017.39 77 Ac-$/r8SQQTFS$/LWRLLAibQN-NH2 817 2066.17 1034.09 1034.80 78 Ac-QSQ$/r8TFSNLW$/LLAibQN-NH2 818 2024.11 1013.06 1014.34 79 Ac-QSQQTFS$/r8LWRLLA$/N-NH2 819 2052.15 1027.08 1027.16 80 Ac-QS$/r5QTFSt//NLW$/LLAibQN-NH2 820 2088.18 1045.09 1047.10 81 Ac-QSQQTFSNLWRLLAibQN-NH2 821 1988.02 995.01 995.31 82 Hep/QSQ$/TF$/r8NLWRLL$/QN-NH2 822 2215.29 1108.65 1108.93 83 Ac-ASQQTF$r8NLRWLL$QN-NH2 823 2051.13 1026.57 1026.90 84 Ac-QSQQTF$/r8NLWRW/Q-NH2 824 2022.14 1012.07 1012.66 85 Ac-QSQQTF$r8NLWRLL$Q-NH2 825 1994.11 998.06 998.42 86 Ac-AAARAA$r8AAARAA$AA-NH2 826 1515.90 758.95 759.21 87 Ac-LTFEHYWAQLTSA-NH2 827 1606.78 804.39 804.59 88 Ac-LTF$r8HYWAQL$SA-NH2 828 1668.90 835.45 835.67 89 Ac-ASQQTFSNLWRLLPQN-NH2 829 1943.00 972.50 973.27 90 Ac-QS$QTFStNLW$r5LLAibQN-NH2 830 2032.12 1017.06 1017.30 91 Ac-QSQQTFAibNLWRLLAibQN-NH2 831 1986.04 994.02 994.19 92 Ac-QSQQTFNleNLWRLLNleQN-NH2 832 2042.11 1022.06 1022.23 93 Ac-QSQQTF$/r8NLWRLLAibQN-NH2 833 2082.14 1042.07 1042.23 94 Ac-QSQQTF$/r8NLWRLLNleQN-NH2 834 2110.17 1056.09 1056.29 95 Ac-QSQQTFAibNLWRLL$/QN-NH2 835 2040.09 1021.05 1021.25 96 Ac-QSQQTFNleNLWRW/QN-NH2 836 2068.12 1035.06 1035.31 97 Ac-QSQQTF%r8NL6clWRNleL%QN-NH2 837 2144.13 1073.07 1073.32 98 Ac-QSQQTF%r8NLMe6clWRLL%QN-NH2 838 2158.15 1080.08 1080.31 101 Ac-FNle$YWE$L-NH2 839 1160.63 — 1161.70 102 Ac-F$r8AYWELL$A-NH2 840 1344.75 — 1345.90 103 Ac-F$r8AYWQLL$A-NH2 841 1343.76 — 1344.83 104 Ac-NlePRF$r8NYWELL$QN-NH2 842 1925.06 963.53 963.69 105 Ac-NlePRF$r8DYWRLL$QN-NH2 843 1953.10 977.55 977.68 106 Ac-NlePRF$r8NYWRLL$Q-NH2 844 1838.07 920.04 920.18 107 Ac-NlePRF$r8NYWRLL$-NH2 845 1710.01 856.01 856.13 108 Ac-QSQQTF$r8DLWRLL$QN-NH2 846 2109.14 1055.57 1055.64 109 Ac-QSQQTF$r8NLWRLL$EN-NH2 847 2109.14 1055.57 1055.70 110 Ac-QSQQTF$r8NLWRLL$QD-NH2 848 2109.14 1055.57 1055.64 111 Ac-QSQQTF$r8NLWRLL$S-NH2 849 1953.08 977.54 977.60 112 Ac-ESQQTF$r8NLWRLL$QN-NH2 850 2109.14 1055.57 1055.70 113 Ac-LTF$r8NLWRNleL$Q-NH2 851 1635.99 819.00 819.10 114 Ac-LRF$r8NLWRNleL$Q-NH2 852 1691.04 846.52 846.68 115 Ac-QSQQTF$r8NWWRNleL$QN-NH2 853 2181.15 1091.58 1091.64 116 Ac-QSQQTF$r8NLWRNleL$Q-NH2 854 1994.11 998.06 998.07 117 Ac-QTF$r8NLWRNleL$QN-NH2 855 1765.00 883.50 883.59 118 Ac-NlePRF$r8NWWRLL$QN-NH2 856 1975.13 988.57 988.75 119 Ac-NlePRF$r8NWWRLL$A-NH2 857 1804.07 903.04 903.08 120 Ac-TSFAEYWNLLNH2 858 1467.70 734.85 734.90 121 Ac-QTF$r8HWWSQL$S-NH2 859 1651.85 826.93 827.12 122 Ac-FM$YWE$L-NH2 860 1178.58 — 1179.64 123 Ac-QTFEHWWSQLLS-NH2 861 1601.76 801.88 801.94 124 Ac-QSQQTF$r8NLAmwRLNle$QN-NH2 862 2122.17 1062.09 1062.24 125 Ac-FMAibY6clWEAc3cL-NH2 863 1130.47 — 1131.53 126 Ac-FNle$Y6clWE$L-NH2 864 1194.59 — 1195.64 127 Ac-F$zr8AY6clWEAc3cL$z-NH2 865 1277.63 639.82 1278.71 128 Ac-F$r8AY6clWEAc3cL$A-NH2 866 1348.66 — 1350.72 129 Ac-NlePRF$r8NY6clWRLL$QN-NH2 867 1986.08 994.04 994.64 130 Ac-AF$r8AAWALA$A-NH2 868 1223.71 — 1224.71 131 Ac-TF$r8AAWRLA$Q-NH2 869 1395.80 698.90 399.04 132 Pr-TF$r8AAWRLA$Q-NH2 870 1409.82 705.91 706.04 133 Ac-QSQQTF%r8NLWRNleL%QN-NH2 871 2110.17 1056.09 1056.22 134 Ac-LTF%r8HYWAQL%SA-NH2 872 1670.92 836.46 836.58 135 Ac-NlePRF%r8NYWRLL%QN-NH2 873 1954.13 978.07 978.19 136 Ac-NlePRF%r8NY6clWRLL%QN-NH2 874 1988.09 995.05 995.68 137 Ac-LTF%r8HY6clWAQL%S-NH2 875 1633.84 817.92 817.93 138 Ac-QS%QTF%StNLWRLL%s8QN-NH2 876 2149.24 1075.62 1075.65 139 Ac-LTF%r8HY6clWRQL%S-NH2 877 1718.91 860.46 860.54 140 Ac-QSQQTF%r8NL6clWRLL%QN-NH2 878 2144.13 1073.07 1073.64 141 Ac-%r8SQQTFS%LWRLLAibQN-NH2 879 2040.15 1021.08 1021.13 142 Ac-LTF%r8HYWAQL%S-NH2 880 1599.88 800.94 801.09 143 Ac-TSF%r8QYWNLL%P-NH2 881 1602.88 802.44 802.58 147 Ac-LTFEHYWAQLTS-NH2 882 1535.74 768.87 769.5 152 Ac-F$er8AY6clWEAc3cL$e-NH2 883 1277.63 639.82 1278.71 153 Ac-AF$r8AAWALA$A-NH2 884 1277.63 639.82 1277.84 154 Ac-TF$r8AAWRLA$Q-NH2 885 1395.80 698.90 699.04 155 Pr-TF$r8AAWRLA$Q-NH2 886 1409.82 705.91 706.04 156 Ac-LTF$er8HYWAQL$eS-NH2 887 1597.87 799.94 800.44 159 Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH2 888 2745.30 1373.65 1372.99 160 Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN- 889 2669.27 1335.64 1336.09 NH2 161 Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH2 890 2589.26 1295.63 1296.2 162 Ac-LTF$/r8HYWAQL$/S-NH2 891 1625.90 813.95 814.18 163 Ac-F%r8HY6clWRAc3cL%-NH2 892 1372.72 687.36 687.59 164 Ac-QTF%r8HWWSQL%S-NH2 893 1653.87 827.94 827.94 165 Ac-LTA$r8HYWRQL$S-NH2 894 1606.90 804.45 804.66 166 Ac-Q$r8QQTFSN$WRLLAibQN-NH2 895 2080.12 1041.06 1041.61 167 Ac-QSQQ$r8FSNLWR$LAibQN-NH2 896 2066.11 1034.06 1034.58 168 Ac-F$r8AYWEAc3cL$A-NH2 897 1314.70 658.35 1315.88 169 Ac-F$r8AYWEAc3cL$S-NH2 898 1330.70 666.35 1331.87 170 Ac-F$r8AYWEAc3cL$Q-NH2 899 1371.72 686.86 1372.72 171 Ac-F$r8AYWEAibL$S-NH2 900 1332.71 667.36 1334.83 172 Ac-F$r8AYWEAL$S-NH2 901 1318.70 660.35 1319.73 173 Ac-F$r8AYWEQL$S-NH2 902 1375.72 688.86 1377.53 174 Ac-F$r8HYWEQL$S-NH2 903 1441.74 721.87 1443.48 175 Ac-F$r8HYWAQL$S-NH2 904 1383.73 692.87 1385.38 176 Ac-F$r8HYWAAc3cL$S-NH2 905 1338.71 670.36 1340.82 177 Ac-F$r8HYWRAc3cL$S-NH2 906 1423.78 712.89 713.04 178 Ac-F$r8AYWEAc3cL#A-NH2 907 1300.69 651.35 1302.78 179 Ac-NlePTF%r8NYWRLL%QN-NH2 908 1899.08 950.54 950.56 180 Ac-TF$r8AAWRAL$Q-NH2 909 1395.80 698.90 699.13 181 Ac-TSF%r8HYWAQL%S-NH2 910 1573.83 787.92 787.98 184 Ac-F%r8AY6clWEAc3cL%A-NH2 911 1350.68 676.34 676.91 185 Ac-LTF$r8HYWAQI$S-NH2 912 1597.87 799.94 800.07 186 Ac-LTF$r8HYWAQNle$S-NH2 913 1597.87 799.94 800.07 187 Ac-LTF$r8HYWAQL$A-NH2 914 1581.87 791.94 792.45 188 Ac-LTF$r8HYWAQL$Abu-NH2 915 1595.89 798.95 799.03 189 Ac-LTF$r8HYWAbuQL$S-NH2 916 1611.88 806.94 807.47 190 Ac-LTF$er8AYWAQL$eS-NH2 917 1531.84 766.92 766.96 191 Ac-LAF$r8HYWAQL$S-NH2 918 1567.86 784.93 785.49 192 Ac-LAF$r8AYWAQL$S-NH2 919 1501.83 751.92 752.01 193 Ac-LTF$er8AYWAQL$eA-NH2 920 1515.85 758.93 758.97 194 Ac-LAF$r8AYWAQL$A-NH2 921 1485.84 743.92 744.05 195 Ac-LTF$r8NLWANleL$Q-NH2 922 1550.92 776.46 776.61 196 Ac-LTF$r8NLWANleL$A-NH2 923 1493.90 747.95 1495.6 197 Ac-LTF$r8ALWANleL$Q-NH2 924 1507.92 754.96 755 198 Ac-LAF$r8NLWANleL$Q-NH2 925 1520.91 761.46 761.96 199 Ac-LAF$r8ALWANleL$A-NH2 926 1420.89 711.45 1421.74 200 Ac-A$r8AYWEAc3cL$A-NH2 927 1238.67 620.34 1239.65 201 Ac-F$r8AYWEAc3cL$AA-NH2 928 1385.74 693.87 1386.64 202 Ac-F$r8AYWEAc3cL$Abu-NH2 929 1328.72 665.36 1330.17 203 Ac-F$r8AYWEAc3cL$Nle-NH2 930 1356.75 679.38 1358.22 204 Ac-F$r5AYWEAc3cL$s8A-NH2 931 1314.70 658.35 1315.51 205 Ac-F$AYWEAc3cL$r8A-NH2 932 1314.70 658.35 1315.66 206 Ac-F$r8AYWEAc3cI$A-NH2 933 1314.70 658.35 1316.18 207 Ac-F$r8AYWEAc3cNle$A-NH2 934 1314.70 658.35 1315.66 208 Ac-F$r8AYWEAmlL$A-NH2 935 1358.76 680.38 1360.21 209 Ac-F$r8AYWENleL$A-NH2 936 1344.75 673.38 1345.71 210 Ac-F$r8AYWQAc3cL$A-NH2 937 1313.72 657.86 1314.7 211 Ac-F$r8AYWAAc3cL$A-NH2 938 1256.70 629.35 1257.56 212 Ac-F$r8AYWAbuAc3cL$A-NH2 939 1270.71 636.36 1272.14 213 Ac-F$r8AYWNleAc3cL$A-NH2 940 1298.74 650.37 1299.67 214 Ac-F$r8AbuYWEAc3cL$A-NH2 941 1328.72 665.36 1329.65 215 Ac-F$r8NleYWEAc3cL$A-NH2 942 1356.75 679.38 1358.66 216 5-FAM-BaLTFEHYWAQLTS-NH2 943 1922.82 962.41 962.87 217 5-FAM-BaLTF%r8HYWAQL%S-NH2 944 1986.96 994.48 994.97 218 Ac-LTF$r8HYWAQhL$S-NH2 945 1611.88 806.94 807 219 Ac-LTF$r8HYWAQTle$S-NH2 946 1597.87 799.94 799.97 220 Ac-LTF$r8HYWAQAdm$S-NH2 947 1675.91 838.96 839.09 221 Ac-LTF$r8HYWAQhCha$S-NH2 948 1651.91 826.96 826.98 222 Ac-LTF$r8HYWAQCha$S-NH2 949 1637.90 819.95 820.02 223 Ac-LTF$r8HYWAc6cQL$S-NH2 950 1651.91 826.96 826.98 224 Ac-LTF$r8HYWAc5cQL$S-NH2 951 1637.90 819.95 820.02 225 Ac-LThF$r8HYWAQL$S-NH2 952 1611.88 806.94 807 226 Ac-LTIgl$r8HYWAQL$S-NH2 953 1625.90 813.95 812.99 227 Ac-LTF$r8HYWAQChg$S-NH2 954 1623.88 812.94 812.99 228 Ac-LTF$r8HYWAQF$S-NH2 955 1631.85 816.93 816.99 229 Ac-LTF$r8HYWAQIgl$S-NH2 956 1659.88 830.94 829.94 230 Ac-LTF$r8HYWAQCba$S-NH2 957 1609.87 805.94 805.96 231 Ac-LTF$r8HYWAQCpg$S-NH2 958 1609.87 805.94 805.96 232 Ac-LTF$r8HhYWAQL$S-NH2 959 1611.88 806.94 807 233 Ac-F$r8AYWEAc3chL$A-NH2 960 1328.72 665.36 665.43 234 Ac-F$r8AYWEAc3cTle$A-NH2 961 1314.70 658.35 1315.62 235 Ac-F$r8AYWEAc3cAdm$A-NH2 962 1392.75 697.38 697.47 236 Ac-F$r8AYWEAc3chCha$A-NH2 963 1368.75 685.38 685.34 237 Ac-F$r8AYWEAc3cCha$A-NH2 964 1354.73 678.37 678.38 238 Ac-F$r8AYWEAc6cL$A-NH2 965 1356.75 679.38 679.42 239 Ac-F$r8AYWEAc5cL$A-NH2 966 1342.73 672.37 672.46 240 Ac-hF$r8AYWEAc3cL$A-NH2 967 1328.72 665.36 665.43 241 Ac-Igl$r8AYWEAc3cL$A-NH2 968 1342.73 672.37 671.5 243 Ac-F$r8AYWEAc3cF$A-NH2 969 1348.69 675.35 675.35 244 Ac-F$r8AYWEAc3cIgl$A-NH2 970 1376.72 689.36 688.37 245 Ac-F$r8AYWEAc3cCba$A-NH2 971 1326.70 664.35 664.47 246 Ac-F$r8AYWEAc3cCpg$A-NH2 972 1326.70 664.35 664.39 247 Ac-F$r8AhYWEAc3cL$A-NH2 973 1328.72 665.36 665.43 248 Ac-F$r8AYWEAc3cL$Q-NH2 974 1371.72 686.86 1372.87 249 Ac-F$r8AYWEAibL$A-NH2 975 1316.72 659.36 1318.18 250 Ac-F$r8AYWEAL$A-NH2 976 1302.70 652.35 1303.75 251 Ac-LAF$r8AYWAAL$A-NH2 977 1428.82 715.41 715.49 252 Ac-LTF$r8HYWAAc3cL$S-NH2 978 1552.84 777.42 777.5 253 Ac-NleTF$r8HYWAQL$S-NH2 979 1597.87 799.94 800.04 254 Ac-VTF$r8HYWAQL$S-NH2 980 1583.85 792.93 793.04 255 Ac-FTF$r8HYWAQL$S-NH2 981 1631.85 816.93 817.02 256 Ac-WTF$r8HYWAQL$S-NH2 982 1670.86 836.43 836.85 257 Ac-RTF$r8HYWAQL$S-NH2 983 1640.88 821.44 821.9 258 Ac-KTF$r8HYWAQL$S-NH2 984 1612.88 807.44 807.91 259 Ac-LNleF$r8HYWAQL$S-NH2 985 1609.90 805.95 806.43 260 Ac-LVF$r8HYWAQL$S-NH2 986 1595.89 798.95 798.93 261 Ac-LFF$r8HYWAQL$S-NH2 987 1643.89 822.95 823.38 262 Ac-LWF$r8HYWAQL$S-NH2 988 1682.90 842.45 842.55 263 Ac-LRF$r8HYWAQL$S-NH2 989 1652.92 827.46 827.52 264 Ac-LKF$r8HYWAQL$S-NH2 990 1624.91 813.46 813.51 265 Ac-LTF$r8NleYWAQL$S-NH2 991 1573.89 787.95 788.05 266 Ac-LTF$r8VYWAQL$S-NH2 992 1559.88 780.94 780.98 267 Ac-LTF$r8FYWAQL$S-NH2 993 1607.88 804.94 805.32 268 Ac-LTF$r8WYWAQL$S-NH2 994 1646.89 824.45 824.86 269 Ac-LTF$r8RYWAQL$S-NH2 995 1616.91 809.46 809.51 270 Ac-LTF$r8KYWAQL$S-NH2 996 1588.90 795.45 795.48 271 Ac-LTF$r8HNleWAQL$S-NH2 997 1547.89 774.95 774.98 272 Ac-LTF$r8HVWAQL$S-NH2 998 1533.87 767.94 767.95 273 Ac-LTF$r8HFWAQL$S-NH2 999 1581.87 791.94 792.3 274 Ac-LTF$r8HWWAQL$S-NH2 1000 1620.88 811.44 811.54 275 Ac-LTF$r8HRWAQL$S-NH2 1001 1590.90 796.45 796.52 276 Ac-LTF$r8HKWAQL$S-NH2 1002 1562.90 782.45 782.53 277 Ac-LTF$r8HYWNleQL$S-NH2 1003 1639.91 820.96 820.98 278 Ac-LTF$r8HYWVQL$S-NH2 1004 1625.90 813.95 814.03 279 Ac-LTF$r8HYWFQL$S-NH2 1005 1673.90 837.95 838.03 280 Ac-LTF$r8HYWWQL$S-NH2 1006 1712.91 857.46 857.5 281 Ac-LTF$r8HYWKQL$S-NH2 1007 1654.92 828.46 828.49 282 Ac-LTF$r8HYWANleL$S-NH2 1008 1582.89 792.45 792.52 283 Ac-LTF$r8HYWAVL$S-NH2 1009 1568.88 785.44 785.49 284 Ac-LTF$r8HYWAFL$S-NH2 1010 1616.88 809.44 809.47 285 Ac-LTF$r8HYWAWL$S-NH2 1011 1655.89 828.95 829 286 Ac-LTF$r8HYWARL$S-NH2 1012 1625.91 813.96 813.98 287 Ac-LTF$r8HYWAQL$Nle-NH2 1013 1623.92 812.96 813.39 288 Ac-LTF$r8HYWAQL$V-NH2 1014 1609.90 805.95 805.99 289 Ac-LTF$r8HYWAQL$F-NH2 1015 1657.90 829.95 830.26 290 Ac-LTF$r8HYWAQL$W-NH2 1016 1696.91 849.46 849.5 291 Ac-LTF$r8HYWAQL$R-NH2 1017 1666.94 834.47 834.56 292 Ac-LTF$r8HYWAQL$K-NH2 1018 1638.93 820.47 820.49 293 Ac-Q$r8QQTFSN$WRLLAibQN-NH2 1019 2080.12 1041.06 1041.54 294 Ac-QSQQ$r8FSNLWR$LAibQN-NH2 1020 2066.11 1034.06 1034.58 295 Ac-LT2Pal$r8HYWAQL$S-NH2 1021 1598.86 800.43 800.49 296 Ac-LT3Pal$r8HYWAQL$S-NH2 1022 1598.86 800.43 800.49 297 Ac-LT4Pal$r8HYWAQL$S-NH2 1023 1598.86 800.43 800.49 298 Ac-LTF2CF3$r8HYWAQL$S-NH2 1024 1665.85 833.93 834.01 299 Ac-LTF2CN$r8HYWAQL$S-NH2 1025 1622.86 812.43 812.47 300 Ac-LTF2Me$r8HYWAQL$S-NH2 1026 1611.88 806.94 807 301 Ac-LTF3Cl$r8HYWAQL$S-NH2 1027 1631.83 816.92 816.99 302 Ac-LTF4CF3$r8HYWAQL$S-NH2 1028 1665.85 833.93 833.94 303 Ac-LTF4tBu$r8HYWAQL$S-NH2 1029 1653.93 827.97 828.02 304 Ac-LTF5F$r8HYWAQL$S-NH2 1030 1687.82 844.91 844.96 305 Ac-LTF$r8HY3BthAAQL$S-NH2 1031 1614.83 808.42 808.48 306 Ac-LTF2Br$r8HYWAQL$S-NH2 1032 1675.78 838.89 838.97 307 Ac-LTF4Br$r8HYWAQL$S-NH2 1033 1675.78 838.89 839.86 308 Ac-LTF2Cl$r8HYWAQL$S-NH2 1034 1631.83 816.92 816.99 309 Ac-LTF4Cl$r8HYWAQL$S-NH2 1035 1631.83 816.92 817.36 310 Ac-LTF3CN$r8HYWAQL$S-NH2 1036 1622.86 812.43 812.47 311 Ac-LTF4CN$r8HYWAQL$S-NH2 1037 1622.86 812.43 812.47 312 Ac-LTF34Cl2$r8HYWAQL$S-NH2 1038 1665.79 833.90 833.94 313 Ac-LTF34F2$r8HYWAQL$S-NH2 1039 1633.85 817.93 817.95 314 Ac-LTF35F2$r8HYWAQL$S-NH2 1040 1633.85 817.93 817.95 315 Ac-LTDip$r8HYWAQL$S-NH2 1041 1673.90 837.95 838.01 316 Ac-LTF2F$r8HYWAQL$S-NH2 1042 1615.86 808.93 809 317 Ac-LTF3F$r8HYWAQL$S-NH2 1043 1615.86 808.93 809 318 Ac-LTF4F$r8HYWAQL$S-NH2 1044 1615.86 808.93 809 319 Ac-LTF4I$r8HYWAQL$S-NH2 1045 1723.76 862.88 862.94 320 Ac-LTF3Me$r8HYWAQL$S-NH2 1046 1611.88 806.94 807.07 321 Ac-LTF4Me$r8HYWAQL$S-NH2 1047 1611.88 806.94 807 322 Ac-LT1Nal$r8HYWAQL$S-NH2 1048 1647.88 824.94 824.98 323 Ac-LT2Nal$r8HYWAQL$S-NH2 1049 1647.88 824.94 825.06 324 Ac-LTF3CF3$r8HYWAQL$S-NH2 1050 1665.85 833.93 834.01 325 Ac-LTF4NO2$r8HYWAQL$S-NH2 1051 1642.85 822.43 822.46 326 Ac-LTF3NO2$r8HYWAQL$S-NH2 1052 1642.85 822.43 822.46 327 Ac-LTF$r82ThiYWAQL$S-NH2 1053 1613.83 807.92 807.96 328 Ac-LTF$r8HBipWAQL$S-NH2 1054 1657.90 829.95 830.01 329 Ac-LTF$r8HF4tBuWAQL$S-NH2 1055 1637.93 819.97 820.02 330 Ac-LTF$r8HF4CF3WAQL$S-NH2 1056 1649.86 825.93 826.02 331 Ac-LTF$r8HF4ClWAQL$S-NH2 1057 1615.83 808.92 809.37 332 Ac-LTF$r8HF4MeWAQL$S-NH2 1058 1595.89 798.95 799.01 333 Ac-LTF$r8HF4BrWAQL$S-NH2 1059 1659.78 830.89 830.98 334 Ac-LTF$r8HF4CNWAQL$S-NH2 1060 1606.87 804.44 804.56 335 Ac-LTF$r8HF4NO2WAQL$S-NH2 1061 1626.86 814.43 814.55 336 Ac-LTF$r8H1NalWAQL$S-NH2 1062 1631.89 816.95 817.06 337 Ac-LTF$r8H2NalWAQL$S-NH2 1063 1631.89 816.95 816.99 338 Ac-LTF$r8HWAQL$S-NH2 1064 1434.80 718.40 718.49 339 Ac-LTF$r8HY1NalAQL$S-NH2 1065 1608.87 805.44 805.52 340 Ac-LTF$r8HY2NalAQL$S-NH2 1066 1608.87 805.44 805.52 341 Ac-LTF$r8HYWAQI$S-NH2 1067 1597.87 799.94 800.07 342 Ac-LTF$r8HYWAQNle$S-NH2 1068 1597.87 799.94 800.44 343 Ac-LTF$er8HYWAQL$eA-NH2 1069 1581.87 791.94 791.98 344 Ac-LTF$r8HYWAQL$Abu-NH2 1070 1595.89 798.95 799.03 345 Ac-LTF$r8HYWAbuQL$S-NH2 1071 1611.88 806.94 804.47 346 Ac-LAF$r8HYWAQL$S-NH2 1072 1567.86 784.93 785.49 347 Ac-LTF$r8NLWANleL$Q-NH2 1073 1550.92 776.46 777.5 348 Ac-LTF$r8ALWANleL$Q-NH2 1074 1507.92 754.96 755.52 349 Ac-LAF$r8NLWANleL$Q-NH2 1075 1520.91 761.46 762.48 350 Ac-F$r8AYWAAc3cL$A-NH2 1076 1256.70 629.35 1257.56 351 Ac-LTF$r8AYWAAL$S-NH2 1077 1474.82 738.41 738.55 352 Ac-LVF$r8AYWAQL$S-NH2 1078 1529.87 765.94 766 353 Ac-LTF$r8AYWAbuQL$S-NH2 1079 1545.86 773.93 773.92 354 Ac-LTF$r8AYWNleQL$S-NH2 1080 1573.89 787.95 788.17 355 Ac-LTF$r8AbuYWAQL$S-NH2 1081 1545.86 773.93 773.99 356 Ac-LTF$r8AYWHQL$S-NH2 1082 1597.87 799.94 799.97 357 Ac-LTF$r8AYWKQL$S-NH2 1083 1588.90 795.45 795.53 358 Ac-LTF$r8AYWOQL$S-NH2 1084 1574.89 788.45 788.5 359 Ac-LTF$r8AYWRQL$S-NH2 1085 1616.91 809.46 809.51 360 Ac-LTF$r8AYWSQL$S-NH2 1086 1547.84 774.92 774.96 361 Ac-LTF$r8AYWRAL$S-NH2 1087 1559.89 780.95 780.95 362 Ac-LTF$r8AYWRQL$A-NH2 1088 1600.91 801.46 801.52 363 Ac-LTF$r8AYWRAL$A-NH2 1089 1543.89 772.95 773.03 364 Ac-LTF$r5HYWAQL$s8S-NH2 1090 1597.87 799.94 799.97 365 Ac-LTF$HYWAQL$r8S-NH2 1091 1597.87 799.94 799.97 366 Ac-LTF$r8HYWAAL$S-NH2 1092 1540.84 771.42 771.48 367 Ac-LTF$r8HYWAAbuL$S-NH2 1093 1554.86 778.43 778.51 368 Ac-LTF$r8HYWALL$S-NH2 1094 1582.89 792.45 792.49 369 Ac-F$r8AYWHAL$A-NH2 1095 1310.72 656.36 656.4 370 Ac-F$r8AYWAAL$A-NH2 1096 1244.70 623.35 1245.61 371 Ac-F$r8AYWSAL$A-NH2 1097 1260.69 631.35 1261.6 372 Ac-F$r8AYWRAL$A-NH2 1098 1329.76 665.88 1330.72 373 Ac-F$r8AYWKAL$A-NH2 1099 1301.75 651.88 1302.67 374 Ac-F$r8AYWOAL$A-NH2 1100 1287.74 644.87 1289.13 375 Ac-F$r8VYWEAc3cL$A-NH2 1101 1342.73 672.37 1343.67 376 Ac-F$r8FYWEAc3cL$A-NH2 1102 1390.73 696.37 1392.14 377 Ac-F$r8WYWEAc3cL$A-NH2 1103 1429.74 715.87 1431.44 378 Ac-F$r8RYWEAc3cL$A-NH2 1104 1399.77 700.89 700.95 379 Ac-F$r8KYWEAc3cL$A-NH2 1105 1371.76 686.88 686.97 380 Ac-F$r8ANleWEAc3cL$A-NH2 1106 1264.72 633.36 1265.59 381 Ac-F$r8AVWEAc3cL$A-NH2 1107 1250.71 626.36 1252.2 382 Ac-F$r8AFWEAc3cL$A-NH2 1108 1298.71 650.36 1299.64 383 Ac-F$r8AWWEAc3cL$A-NH2 1109 1337.72 669.86 1338.64 384 Ac-F$r8ARWEAc3cL$A-NH2 1110 1307.74 654.87 655 385 Ac-F$r8AKWEAc3cL$A-NH2 1111 1279.73 640.87 641.01 386 Ac-F$r8AYWVAc3cL$A-NH2 1112 1284.73 643.37 643.38 387 Ac-F$r8AYWFAc3cL$A-NH2 1113 1332.73 667.37 667.43 388 Ac-F$r8AYWWAc3cL$A-NH2 1114 1371.74 686.87 686.97 389 Ac-F$r8AYWRAc3cL$A-NH2 1115 1341.76 671.88 671.94 390 Ac-F$r8AYWKAc3cL$A-NH2 1116 1313.75 657.88 657.88 391 Ac-F$r8AYWEVL$A-NH2 1117 1330.73 666.37 666.47 392 Ac-F$r8AYWEFL$A-NH2 1118 1378.73 690.37 690.44 393 Ac-F$r8AYWEWL$A-NH2 1119 1417.74 709.87 709.91 394 Ac-F$r8AYWERL$A-NH2 1120 1387.77 694.89 1388.66 395 Ac-F$r8AYWEKL$A-NH2 1121 1359.76 680.88 1361.21 396 Ac-F$r8AYWEAc3cL$V-NH2 1122 1342.73 672.37 1343.59 397 Ac-F$r8AYWEAc3cL$F-NH2 1123 1390.73 696.37 1392.58 398 Ac-F$r8AYWEAc3cL$W-NH2 1124 1429.74 715.87 1431.29 399 Ac-F$r8AYWEAc3cL$R-NH2 1125 1399.77 700.89 700.95 400 Ac-F$r8AYWEAc3cL$K-NH2 1126 1371.76 686.88 686.97 401 Ac-F$r8AYWEAc3cL$AV-NH2 1127 1413.77 707.89 707.91 402 Ac-F$r8AYWEAc3cL$AF-NH2 1128 1461.77 731.89 731.96 403 Ac-F$r8AYWEAc3cL$AW-NH2 1129 1500.78 751.39 751.5 404 Ac-F$r8AYWEAc3cL$AR-NH2 1130 1470.80 736.40 736.47 405 Ac-F$r8AYWEAc3cL$AK-NH2 1131 1442.80 722.40 722.41 406 Ac-F$r8AYWEAc3cL$AH-NH2 1132 1451.76 726.88 726.93 407 Ac-LTF2NO2$r8HYWAQL$S-NH2 1133 1642.85 822.43 822.54 408 Ac-LTA$r8HYAAQL$S-NH2 1134 1406.79 704.40 704.5 409 Ac-LTF$r8HYAAQL$S-NH2 1135 1482.82 742.41 742.47 410 Ac-QSQQTF$r8NLWALL$AN-NH2 1136 1966.07 984.04 984.38 411 Ac-QAibQQTF$r8NLWALL$AN-NH2 1137 1964.09 983.05 983.42 412 Ac-QAibQQTF$r8ALWALL$AN-NH2 1138 1921.08 961.54 961.59 413 Ac-AAAATF$r8AAWAAL$AA-NH2 1139 1608.90 805.45 805.52 414 Ac-F$r8AAWRAL$Q-NH2 1140 1294.76 648.38 648.48 415 Ac-TF$r8AAWAAL$Q-NH2 1141 1310.74 656.37 1311.62 416 Ac-TF$r8AAWRAL$A-NH2 1142 1338.78 670.39 670.46 417 Ac-VF$r8AAWRAL$Q-NH2 1143 1393.82 697.91 697.99 418 Ac-AF$r8AAWAAL$A-NH2 1144 1223.71 612.86 1224.67 420 Ac-TF$r8AAWKAL$Q-NH2 1145 1367.80 684.90 684.97 421 Ac-TF$r8AAWOAL$Q-NH2 1146 1353.78 677.89 678.01 422 Ac-TF$r8AAWSAL$Q-NH2 1147 1326.73 664.37 664.47 423 Ac-LTF$r8AAWRAL$Q-NH2 1148 1508.89 755.45 755.49 424 Ac-F$r8AYWAQL$A-NH2 1149 1301.72 651.86 651.96 425 Ac-F$r8AWWAAL$A-NH2 1150 1267.71 634.86 634.87 426 Ac-F$r8AWWAQL$A-NH2 1151 1324.73 663.37 663.43 427 Ac-F$r8AYWEAL$-NH2 1152 1231.66 616.83 1232.93 428 Ac-F$r8AYWAAL$-NH2 1153 1173.66 587.83 1175.09 429 Ac-F$r8AYWKAL$-NH2 1154 1230.72 616.36 616.44 430 Ac-F$r8AYWOAL$-NH2 1155 1216.70 609.35 609.48 431 Ac-F$r8AYWQAL$-NH2 1156 1230.68 616.34 616.44 432 Ac-F$r8AYWAQL$-NH2 1157 1230.68 616.34 616.37 433 Ac-F$r8HYWDQL$S-NH2 1158 1427.72 714.86 714.86 434 Ac-F$r8HFWEQL$S-NH2 1159 1425.74 713.87 713.98 435 Ac-F$r8AYWHQL$S-NH2 1160 1383.73 692.87 692.96 436 Ac-F$r8AYWKQL$S-NH2 1161 1374.77 688.39 688.45 437 Ac-F$r8AYWOQL$S-NH2 1162 1360.75 681.38 681.49 438 Ac-F$r8HYWSQL$S-NH2 1163 1399.73 700.87 700.95 439 Ac-F$r8HWWEQL$S-NH2 1164 1464.76 733.38 733.44 440 Ac-F$r8HWWAQL$S-NH2 1165 1406.75 704.38 704.43 441 Ac-F$r8AWWHQL$S-NH2 1166 1406.75 704.38 704.43 442 Ac-F$r8AWWKQL$S-NH2 1167 1397.79 699.90 699.92 443 Ac-F$r8AWWOQL$S-NH2 1168 1383.77 692.89 692.96 444 Ac-F$r8HWWSQL$S-NH2 1169 1422.75 712.38 712.42 445 Ac-LTF$r8NYWANleL$Q-NH2 1170 1600.90 801.45 801.52 446 Ac-LTF$r8NLWAQL$Q-NH2 1171 1565.90 783.95 784.06 447 Ac-LTF$r8NYWANleL$A-NH2 1172 1543.88 772.94 773.03 448 Ac-LTF$r8NLWAQL$A-NH2 1173 1508.88 755.44 755.49 449 Ac-LTF$r8AYWANleL$Q-NH2 1174 1557.90 779.95 780.06 450 Ac-LTF$r8ALWAQL$Q-NH2 1175 1522.89 762.45 762.45 451 Ac-LAF$r8NYWANleL$Q-NH2 1176 1570.89 786.45 786.5 452 Ac-LAF$r8NLWAQL$Q-NH2 1177 1535.89 768.95 769.03 453 Ac-LAF$r8AYWANleL$A-NH2 1178 1470.86 736.43 736.47 454 Ac-LAF$r8ALWAQL$A-NH2 1179 1435.86 718.93 719.01 455 Ac-LAF$r8AYWAAL$A-NH2 1180 1428.82 715.41 715.41 456 Ac-F$r8AYWEAc3cL$AAib-NH2 1181 1399.75 700.88 700.95 457 Ac-F$r8AYWAQL$AA-NH2 1182 1372.75 687.38 687.78 458 Ac-F$r8AYWAAc3cL$AA-NH2 1183 1327.73 664.87 664.84 459 Ac-F$r8AYWSAc3cL$AA-NH2 1184 1343.73 672.87 672.9 460 Ac-F$r8AYWEAc3cL$AS-NH2 1185 1401.73 701.87 701.84 461 Ac-F$r8AYWEAc3cL$AT-NH2 1186 1415.75 708.88 708.87 462 Ac-F$r8AYWEAc3cL$AL-NH2 1187 1427.79 714.90 714.94 463 Ac-F$r8AYWEAc3cL$AQ-NH2 1188 1442.76 722.38 722.41 464 Ac-F$r8AFWEAc3cL$AA-NH2 1189 1369.74 685.87 685.93 465 Ac-F$r8AWWEAc3cL$AA-NH2 1190 1408.75 705.38 705.39 466 Ac-F$r8AYWEAc3cL$SA-NH2 1191 1401.73 701.87 701.99 467 Ac-F$r8AYWEAL$AA-NH2 1192 1373.74 687.87 687.93 468 Ac-F$r8AYWENleL$AA-NH2 1193 1415.79 708.90 708.94 469 Ac-F$r8AYWEAc3cL$AbuA-NH2 1194 1399.75 700.88 700.95 470 Ac-F$r8AYWEAc3cL$NleA-NH2 1195 1427.79 714.90 714.86 471 Ac-F$r8AYWEAibL$NleA-NH2 1196 1429.80 715.90 715.97 472 Ac-F$r8AYWEAL$NleA-NH2 1197 1415.79 708.90 708.94 473 Ac-F$r8AYWENleL$NleA-NH2 1198 1457.83 729.92 729.96 474 Ac-F$r8AYWEAibL$Abu-NH2 1199 1330.73 666.37 666.39 475 Ac-F$r8AYWENleL$Abu-NH2 1200 1358.76 680.38 680.39 476 Ac-F$r8AYWEAL$Abu-NH2 1201 1316.72 659.36 659.36 477 Ac-LTF$r8AFWAQL$S-NH2 1202 1515.85 758.93 759.12 478 Ac-LTF$r8AWWAQL$S-NH2 1203 1554.86 778.43 778.51 479 Ac-LTF$r8AYWAQI$S-NH2 1204 1531.84 766.92 766.96 480 Ac-LTF$r8AYWAQNle$S-NH2 1205 1531.84 766.92 766.96 481 Ac-LTF$r8AYWAQL$SA-NH2 1206 1602.88 802.44 802.48 482 Ac-LTF$r8AWWAQL$A-NH2 1207 1538.87 770.44 770.89 483 Ac-LTF$r8AYWAQI$A-NH2 1208 1515.85 758.93 759.42 484 Ac-LTF$r8AYWAQNle$A-NH2 1209 1515.85 758.93 759.42 485 Ac-LTF$r8AYWAQL$AA-NH2 1210 1586.89 794.45 794.94 486 Ac-LTF$r8HWWAQL$S-NH2 1211 1620.88 811.44 811.47 487 Ac-LTF$r8HRWAQL$S-NH2 1212 1590.90 796.45 796.52 488 Ac-LTF$r8HKWAQL$S-NH2 1213 1562.90 782.45 782.53 489 Ac-LTF$r8HYWAQL$W-NH2 1214 1696.91 849.46 849.5 491 Ac-F$r8AYWAbuAL$A-NH2 1215 1258.71 630.36 630.5 492 Ac-F$r8AbuYWEAL$A-NH2 1216 1316.72 659.36 659.51 493 Ac-NlePRF%r8NYWRLL%QN-NH2 1217 1954.13 978.07 978.54 494 Ac-TSF%r8HYWAQL%S-NH2 1218 1573.83 787.92 787.98 495 Ac-LTF%r8AYWAQL%S-NH2 1219 1533.86 767.93 768 496 Ac-HTF$r8HYWAQL$S-NH2 1220 1621.84 811.92 811.96 497 Ac-LHF$r8HYWAQL$S-NH2 1221 1633.88 817.94 818.02 498 Ac-LTF$r8HHWAQL$S-NH2 1222 1571.86 786.93 786.94 499 Ac-LTF$r8HYWHQL$S-NH2 1223 1663.89 832.95 832.38 500 Ac-LTF$r8HYWAHL$S-NH2 1224 1606.87 804.44 804.48 501 Ac-LTF$r8HYWAQL$H-NH2 1225 1647.89 824.95 824.98 502 Ac-LTF$r8HYWAQL$S-NHPr 1226 1639.91 820.96 820.98 503 Ac-LTF$r8HYWAQL$S-NHsBu 1227 1653.93 827.97 828.02 504 Ac-LTF$r8HYWAQL$S-NHiBu 1228 1653.93 827.97 828.02 505 Ac-LTF$r8HYWAQL$S-NHBn 1229 1687.91 844.96 844.44 506 Ac-LTF$r8HYWAQL$S-NHPe 1230 1700.92 851.46 851.99 507 Ac-LTF$r8HYWAQL$S-NHChx 1231 1679.94 840.97 841.04 508 Ac-ETF$r8AYWAQL$S-NH2 1232 1547.80 774.90 774.96 509 Ac-STF$r8AYWAQL$S-NH2 1233 1505.79 753.90 753.94 510 Ac-LEF$r8AYWAQL$S-NH2 1234 1559.84 780.92 781.25 511 Ac-LSF$r8AYWAQL$S-NH2 1235 1517.83 759.92 759.93 512 Ac-LTF$r8EYWAQL$S-NH2 1236 1589.85 795.93 795.97 513 Ac-LTF$r8SYWAQL$S-NH2 1237 1547.84 774.92 774.96 514 Ac-LTF$r8AYWEQL$S-NH2 1238 1589.85 795.93 795.9 515 Ac-LTF$r8AYWAEL$S-NH2 1239 1532.83 767.42 766.96 516 Ac-LTF$r8AYWASL$S-NH2 1240 1490.82 746.41 746.46 517 Ac-LTF$r8AYWAQL$E-NH2 1241 1573.85 787.93 787.98 518 Ac-LTF2CN$r8HYWAQL$S-NH2 1242 1622.86 812.43 812.47 519 Ac-LTF3Cl$r8HYWAQL$S-NH2 1243 1631.83 816.92 816.99 520 Ac-LTDip$r8HYWAQL$S-NH2 1244 1673.90 837.95 838.01 521 Ac-LTF$r8HYWAQT1e$S-NH2 1245 1597.87 799.94 800.04 522 Ac-F$r8AY6clWEAL$A-NH2 1246 1336.66 669.33 1338.56 523 Ac-F$r8AYdl6brWEAL$A-NH2 1247 1380.61 691.31 692.2 524 Ac-F$r8AYdl6fWEAL$A-NH2 1248 1320.69 661.35 1321.61 525 Ac-F$r8AYdl4mWEAL$A-NH2 1249 1316.72 659.36 659.36 526 Ac-F$r8AYdl5clWEAL$A-NH2 1250 1336.66 669.33 669.35 527 Ac-F$r8AYdl7mWEAL$A-NH2 1251 1316.72 659.36 659.36 528 Ac-LTF%r8HYWAQL%A-NH2 1252 1583.89 792.95 793.01 529 Ac-LTF$r8HCouWAQL$S-NH2 1253 1679.87 840.94 841.38 530 Ac-LTFEHCouWAQLTS-NH2 1254 1617.75 809.88 809.96 531 Ac-LTA$r8HCouWAQL$S-NH2 1255 1603.84 802.92 803.36 532 Ac-F$r8AYWEAL$AbuA-NH2 1256 1387.75 694.88 694.88 533 Ac-F$r8AYWEAl$AA-NH2 1257 1373.74 687.87 687.93 534 Ac-F$r8AYWEANle$AA-NH2 1258 1373.74 687.87 687.93 535 Ac-F$r8AYWEAmlL$AA-NH2 1259 1429.80 715.90 715.97 536 Ac-F$r8AYWQAL$AA-NH2 1260 1372.75 687.38 687.48 537 Ac-F$r8AYWAAL$AA-NH2 1261 1315.73 658.87 658.92 538 Ac-F$r8AYWAbuAL$AA-NH2 1262 1329.75 665.88 665.95 539 Ac-F$r8AYWNleAL$AA-NH2 1263 1357.78 679.89 679.94 540 Ac-F$r8AbuYWEAL$AA-NH2 1264 1387.75 694.88 694.96 541 Ac-F$r8NleYWEAL$AA-NH2 1265 1415.79 708.90 708.94 542 Ac-F$r8FYWEAL$AA-NH2 1266 1449.77 725.89 725.97 543 Ac-LTF$r8HYWAQhL$S-NH2 1267 1611.88 806.94 807 544 Ac-LTF$r8HYWAQAdm$S-NH2 1268 1675.91 838.96 839.04 545 Ac-LTF$r8HYWAQIgl$S-NH2 1269 1659.88 830.94 829.94 546 Ac-F$r8AYWAQL$AA-NH2 1270 1372.75 687.38 687.48 547 Ac-LTF$r8ALWAQL$Q-NH2 1271 1522.89 762.45 762.52 548 Ac-F$r8AYWEAL$AA-NH2 1272 1373.74 687.87 687.93 549 Ac-F$r8AYWENleL$AA-NH2 1273 1415.79 708.90 708.94 550 Ac-F$r8AYWEAibL$Abu-NH2 1274 1330.73 666.37 666.39 551 Ac-F$r8AYWENleL$Abu-NH2 1275 1358.76 680.38 680.38 552 Ac-F$r8AYWEAL$Abu-NH2 1276 1316.72 659.36 659.36 553 Ac-F$r8AYWEAc3cL$AbuA-NH2 1277 1399.75 700.88 700.95 554 Ac-F$r8AYWEAc3cL$NleA-NH2 1278 1427.79 714.90 715.01 555 H-LTF$r8AYWAQL$S-NH2 1279 1489.83 745.92 745.95 556 mdPEG3-LTF$r8AYWAQL$S-NH2 1280 1679.92 840.96 840.97 557 mdPEG7-LTF$r8AYWAQL$S-NH2 1281 1856.02 929.01 929.03 558 Ac-F$r8ApmpEt6clWEAL$A-NH2 1282 1470.71 736.36 788.17 559 Ac-LTF3Cl$r8AYWAQL$S-NH2 1283 1565.81 783.91 809.18 560 Ac-LTF3Cl$r8HYWAQL$A-NH2 1284 1615.83 808.92 875.24 561 Ac-LTF3Cl$r8HYWWQL$S-NH2 1285 1746.87 874.44 841.65 562 Ac-LTF3Cl$r8AYWWQL$S-NH2 1286 1680.85 841.43 824.63 563 Ac-LTF$r8AYWWQL$S-NH2 1287 1646.89 824.45 849.98 564 Ac-LTF$r8HYWWQL$A-NH2 1288 1696.91 849.46 816.67 565 Ac-LTF$r8AYWWQL$A-NH2 1289 1630.89 816.45 776.15 566 Ac-LTF4F$r8AYWAQL$S-NH2 1290 1549.83 775.92 776.15 567 Ac-LTF2F$r8AYWAQL$S-NH2 1291 1549.83 775.92 776.15 568 Ac-LTF3F$r8AYWAQL$S-NH2 1292 1549.83 775.92 785.12 569 Ac-LTF34F2$r8AYWAQL$S-NH2 1293 1567.83 784.92 785.12 570 Ac-LTF35F2$r8AYWAQL$S-NH2 1294 1567.83 784.92 1338.74 571 Ac-F3Cl$r8AYWEAL$A-NH2 1295 1336.66 669.33 705.28 572 Ac-F3Cl$r8AYWEAL$AA-NH2 1296 1407.70 704.85 680.11 573 Ac-F$r8AY6clWEAL$AA-NH2 1297 1407.70 704.85 736.83 574 Ac-F$r8AY6clWEAL$-NH2 1298 1265.63 633.82 784.1 575 Ac-LTF$r8HYWAQLSt/S-NH2 1299 16.03 9.02 826.98 576 Ac-LTF$r8HYWAQL$S-NHsBu 1300 1653.93 827.97 828.02 577 Ac-STF$r8AYWAQL$S-NH2 1301 1505.79 753.90 753.94 578 Ac-LTF$r8AYWAEL$S-NH2 1302 1532.83 767.42 767.41 579 Ac-LTF$r8AYWAQL$E-NH2 1303 1573.85 787.93 787.98 580 mdPEG3-LTF$r8AYWAQL$S-NH2 1304 1679.92 840.96 840.97 581 Ac-LTF$r8AYWAQhL$S-NH2 1305 1545.86 773.93 774.31 583 Ac-LTF$r8AYWAQCha$S-NH2 1306 1571.88 786.94 787.3 584 Ac-LTF$r8AYWAQChg$S-NH2 1307 1557.86 779.93 780.4 585 Ac-LTF$r8AYWAQCba$S-NH2 1308 1543.84 772.92 780.13 586 Ac-LTF$r8AYWAQF$S-NH2 1309 1565.83 783.92 784.2 587 Ac-LTF4F$r8HYWAQhL$S-NH2 1310 1629.87 815.94 815.36 588 Ac-LTF4F$r8HYWAQCha$S-NH2 1311 1655.89 828.95 828.39 589 Ac-LTF4F$r8HYWAQChg$S-NH2 1312 1641.87 821.94 821.35 590 Ac-LTF4F$r8HYWAQCba$S-NH2 1313 1627.86 814.93 814.32 591 Ac-LTF4F$r8AYWAQhL$S-NH2 1314 1563.85 782.93 782.36 592 Ac-LTF4F$r8AYWAQCha$S-NH2 1315 1589.87 795.94 795.38 593 Ac-LTF4F$r8AYWAQChg$S-NH2 1316 1575.85 788.93 788.35 594 Ac-LTF4F$r8AYWAQCba$S-NH2 1317 1561.83 781.92 781.39 595 Ac-LTF3Cl$r8AYWAQhL$S-NH2 1318 1579.82 790.91 790.35 596 Ac-LTF3Cl$r8AYWAQCha$S-NH2 1319 1605.84 803.92 803.67 597 Ac-LTF3Cl$r8AYWAQChg$S-NH2 1320 1591.82 796.91 796.34 598 Ac-LTF3Cl$r8AYWAQCba$S-NH2 1321 1577.81 789.91 789.39 599 Ac-LTF$r8AYWAQhF$S-NH2 1322 1579.84 790.92 791.14 600 Ac-LTF$r8AYWAQF3CF3$S-NH2 1323 1633.82 817.91 818.15 601 Ac-LTF$r8AYWAQF3Me$S-NH2 1324 1581.86 791.93 791.32 602 Ac-LTF$r8AYWAQ1Nal$S-NH2 1325 1615.84 808.92 809.18 603 Ac-LTF$r8AYWAQBip$S-NH2 1326 1641.86 821.93 822.13 604 Ac-LTF$r8FYWAQL$A-NH2 1327 1591.88 796.94 797.33 605 Ac-LTF$r8HYWAQL$S-NHAm 1328 1667.94 834.97 835.92 606 Ac-LTF$r8HYWAQL$S-NHiAm 1329 1667.94 834.97 835.55 607 Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1330 1715.94 858.97 859.79 608 Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1331 1681.96 841.98 842.49 610 Ac-LTF$r8HYWAQL$S-NHnPr 1332 1639.91 820.96 821.58 611 Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1333 1707.98 854.99 855.35 612 Ac-LTF$r8HYWAQL$S-NHHex 1334 1681.96 841.98 842.4 613 Ac-LTF$r8AYWAQL$S-NHmdPeg2 1335 1633.91 817.96 818.35 614 Ac-LTF$r8AYWAQL$A-NHmdPeg2 1336 1617.92 809.96 810.3 615 Ac-LTF$r8AYWAQL$A-NHmdPeg4 1337 1705.97 853.99 854.33 616 Ac-F$r8AYdl4mWEAL$A-NH2 1338 1316.72 659.36 659.44 617 Ac-F$r8AYdl5clWEAL$A-NH2 1339 1336.66 669.33 669.43 618 Ac-LThF$r8AYWAQL$S-NH2 1340 1545.86 773.93 774.11 619 Ac-LT2Nal$r8AYWAQL$S-NH2 1341 1581.86 791.93 792.43 620 Ac-LTA$r8AYWAQL$S-NH2 1342 1455.81 728.91 729.15 621 Ac-LTF$r8AYWVQL$S-NH2 1343 1559.88 780.94 781.24 622 Ac-LTF$r8HYWAAL$A-NH2 1344 1524.85 763.43 763.86 623 Ac-LTF$r8VYWAQL$A-NH2 1345 1543.88 772.94 773.37 624 Ac-LTF$r8IYWAQL$S-NH2 1346 1573.89 787.95 788.17 625 Ac-FTF$r8VYWSQL$S-NH2 1347 1609.85 805.93 806.22 626 Ac-ITF$r8FYWAQL$S-NH2 1348 1607.88 804.94 805.2 627 Ac-2NalTF$r8VYWSQL$S-NH2 1349 1659.87 830.94 831.2 628 Ac-ITF$r8LYWSQL$S-NH2 1350 1589.89 795.95 796.13 629 Ac-FTF$r8FYWAQL$S-NH2 1351 1641.86 821.93 822.13 630 Ac-WTF$r8VYWAQL$S-NH2 1352 1632.87 817.44 817.69 631 Ac-WTF$r8WYWAQL$S-NH2 1353 1719.88 860.94 861.36 632 Ac-VTF$r8AYWSQL$S-NH2 1354 1533.82 767.91 768.19 633 Ac-WTF$r8FYWSQL$S-NH2 1355 1696.87 849.44 849.7 634 Ac-FTF$r8IYWAQL$S-NH2 1356 1607.88 804.94 805.2 635 Ac-WTF$r8VYWSQL$S-NH2 1357 1648.87 825.44 824.8 636 Ac-FTF$r8LYWSQL$S-NH2 1358 1623.87 812.94 812.8 637 Ac-YTF$r8FYWSQL$S-NH2 1359 1673.85 837.93 837.8 638 Ac-LTF$r8AY6clWEAL$A-NH2 1360 1550.79 776.40 776.14 639 Ac-LTF$r8AY6clWSQL$S-NH2 1361 1581.80 791.90 791.68 640 Ac-F$r8AY6clWSAL$A-NH2 1362 1294.65 648.33 647.67 641 Ac-F$r8AY6clWQAL$AA-NH2 1363 1406.72 704.36 703.84 642 Ac-LHF$r8AYWAQL$S-NH2 1364 1567.86 784.93 785.21 643 Ac-LTF$r8AYWAQL$S-NH2 1365 1531.84 766.92 767.17 644 Ac-LTF$r8AHWAQL$S-NH2 1366 1505.84 753.92 754.13 645 Ac-LTF$r8AYWAHL$S-NH2 1367 1540.84 771.42 771.61 646 Ac-LTF$r8AYWAQL$H-NH2 1368 1581.87 791.94 792.15 647 H-LTF$r8AYWAQL$A-NH2 1369 1473.84 737.92 737.29 648 Ac-HHF$r8AYWAQL$S-NH2 1370 1591.83 796.92 797.35 649 Ac-aAibWTF$r8VYWSQL$S-NH2 1371 1804.96 903.48 903.64 650 Ac-AibWTF$r8HYWAQL$S-NH2 1372 1755.91 878.96 879.4 651 Ac-AibAWTF$r8HYWAQL$S-NH2 1373 1826.95 914.48 914.7 652 Ac-fWTF$r8HYWAQL$S-NH2 1374 1817.93 909.97 910.1 653 Ac-AibWWTF$r8HYWAQL$S-NH2 1375 1941.99 972.00 972.2 654 Ac-WTF$r8LYWSQL$S-NH2 1376 1662.88 832.44 832.8 655 Ac-WTF$r8NleYWSQL$S-NH2 1377 1662.88 832.44 832.6 656 Ac-LTF$r8AYWSQL$a-NH2 1378 1531.84 766.92 767.2 657 Ac-LTF$r8EYWARL$A-NH2 1379 1601.90 801.95 802.1 658 Ac-LTF$r8EYWAHL$A-NH2 1380 1582.86 792.43 792.6 659 Ac-aTF$r8AYWAQL$S-NH2 1381 1489.80 745.90 746.08 660 Ac-AibTF$r8AYWAQL$S-NH2 1382 1503.81 752.91 753.11 661 Ac-AmfTF$r8AYWAQL$S-NH2 1383 1579.84 790.92 791.14 662 Ac-AmwTF$r8AYWAQL$S-NH2 1384 1618.86 810.43 810.66 663 Ac-NmLTF$r8AYWAQL$S-NH2 1385 1545.86 773.93 774.11 664 Ac-LNmTF$r8AYWAQL$S-NH2 1386 1545.86 773.93 774.11 665 Ac-LSarF$r8AYWAQL$S-NH2 1387 1501.83 751.92 752.18 667 Ac-LGF$r8AYWAQL$S-NH2 1388 1487.82 744.91 745.15 668 Ac-LTNmF$r8AYWAQL$S-NH2 1389 1545.86 773.93 774.2 669 Ac-TF$r8AYWAQL$S-NH2 1390 1418.76 710.38 710.64 670 Ac-ETF$r8AYWAQL$A-NH2 1391 1531.81 766.91 767.2 671 Ac-LTF$r8EYWAQL$A-NH2 1392 1573.85 787.93 788.1 672 Ac-LT2Nal$r8AYWSQL$S-NH2 1393 1597.85 799.93 800.4 673 Ac-LTF$r8AYWAAL$S-NH2 1394 1474.82 738.41 738.68 674 Ac-LTF$r8AYWAQhCha$S-NH2 1395 1585.89 793.95 794.19 675 Ac-LTF$r8AYWAQChg$S-NH2 1396 1557.86 779.93 780.97 676 Ac-LTF$r8AYWAQCba$S-NH2 1397 1543.84 772.92 773.19 677 Ac-LTF$r8AYWAQF3CF3$S-NH2 1398 1633.82 817.91 818.15 678 Ac-LTF$r8AYWAQ1Nal$S-NH2 1399 1615.84 808.92 809.18 679 Ac-LTF$r8AYWAQBip$S-NH2 1400 1641.86 821.93 822.32 680 Ac-LT2Nal$r8AYWAQL$S-NH2 1401 1581.86 791.93 792.15 681 Ac-LTF$r8AYWVQL$S-NH2 1402 1559.88 780.94 781.62 682 Ac-LTF$r8AWWAQL$S-NH2 1403 1554.86 778.43 778.65 683 Ac-FTF$r8VYWSQL$S-NH2 1404 1609.85 805.93 806.12 684 Ac-ITF$r8FYWAQL$S-NH2 1405 1607.88 804.94 805.2 685 Ac-ITF$r8LYWSQL$S-NH2 1406 1589.89 795.95 796.22 686 Ac-FTF$r8FYWAQL$S-NH2 1407 1641.86 821.93 822.41 687 Ac-VTF$r8AYWSQL$S-NH2 1408 1533.82 767.91 768.19 688 Ac-LTF$r8AHWAQL$S-NH2 1409 1505.84 753.92 754.31 689 Ac-LTF$r8AYWAQL$H-NH2 1410 1581.87 791.94 791.94 690 Ac-LTF$r8AYWAHL$S-NH2 1411 1540.84 771.42 771.61 691 Ac-aAibWTF$r8VYWSQL$S-NH2 1412 1804.96 903.48 903.9 692 Ac-AibWTF$r8HYWAQL$S-NH2 1413 1755.91 878.96 879.5 693 Ac-AibAWTF$r8HYWAQL$S-NH2 1414 1826.95 914.48 914.7 694 Ac-fWTF$r8HYWAQL$S-NH2 1415 1817.93 909.97 910.2 695 Ac-AibWWTF$r8HYWAQL$S-NH2 1416 1941.99 972.00 972.7 696 Ac-WTF$r8LYWSQL$S-NH2 1417 1662.88 832.44 832.7 697 Ac-WTF$r8NleYWSQL$S-NH2 1418 1662.88 832.44 832.7 698 Ac-LTF$r8AYWSQL$a-NH2 1419 1531.84 766.92 767.2 699 Ac-LTF$r8EYWARL$A-NH2 1420 1601.90 801.95 802.2 700 Ac-LTF$r8EYWAHL$A-NH2 1421 1582.86 792.43 792.6 701 Ac-aTF$r8AYWAQL$S-NH2 1422 1489.80 745.90 746.1 702 Ac-AibTF$r8AYWAQL$S-NH2 1423 1503.81 752.91 753.2 703 Ac-AmfTF$r8AYWAQL$S-NH2 1424 1579.84 790.92 791.2 704 Ac-AmwTF$r8AYWAQL$S-NH2 1425 1618.86 810.43 810.7 705 Ac-NmLTF$r8AYWAQL$S-NH2 1426 1545.86 773.93 774.1 706 Ac-LNmTF$r8AYWAQL$S-NH2 1427 1545.86 773.93 774.4 707 Ac-LSarF$r8AYWAQL$S-NH2 1428 1501.83 751.92 752.1 708 Ac-TF$r8AYWAQL$S-NH2 1429 1418.76 710.38 710.8 709 Ac-ETF$r8AYWAQL$A-NH2 1430 1531.81 766.91 767.4 710 Ac-LTF$r8EYWAQL$A-NH2 1431 1573.85 787.93 788.2 711 Ac-WTF$r8VYWSQL$S-NH2 1432 1648.87 825.44 825.2 713 Ac-YTF$r8FYWSQL$S-NH2 1433 1673.85 837.93 837.3 714 Ac-F$r8AY6clWSAL$A-NH2 1434 1294.65 648.33 647.74 715 Ac-ETF$r8EYWVQL$S-NH2 1435 1633.84 817.92 817.36 716 Ac-ETF$r8EHWAQL$A-NH2 1436 1563.81 782.91 782.36 717 Ac-ITF$r8EYWAQL$S-NH2 1437 1589.85 795.93 795.38 718 Ac-ITF$r8EHWVQL$A-NH2 1438 1575.88 788.94 788.42 719 Ac-ITF$r8EHWAQL$S-NH2 1439 1563.85 782.93 782.43 720 Ac-LTF4F$r8AYWAQCba$S-NH2 1440 1561.83 781.92 781.32 721 Ac-LTF3Cl$r8AYWAQhL$S-NH2 1441 1579.82 790.91 790.64 722 Ac-LTF3Cl$r8AYWAQCha$S-NH2 1442 1605.84 803.92 803.37 723 Ac-LTF3Cl$r8AYWAQChg$S-NH2 1443 1591.82 796.91 796.27 724 Ac-LTF3Cl$r8AYWAQCba$S-NH2 1444 1577.81 789.91 789.83 725 Ac-LTF$r8AY6clWSQL$S-NH2 1445 1581.80 791.90 791.75 726 Ac-LTF4F$r8HYWAQhL$S-NH2 1446 1629.87 815.94 815.36 727 Ac-LTF4F$r8HYWAQCba$S-NH2 1447 1627.86 814.93 814.32 728 Ac-LTF4F$r8AYWAQhL$S-NH2 1448 1563.85 782.93 782.36 729 Ac-LTF4F$r8AYWAQChg$S-NH2 1449 1575.85 788.93 788.35 730 Ac-ETF$r8EYWVAL$S-NH2 1450 1576.82 789.41 788.79 731 Ac-ETF$r8EHWAAL$A-NH2 1451 1506.79 754.40 754.8 732 Ac-ITF$r8EYWAAL$S-NH2 1452 1532.83 767.42 767.75 733 Ac-ITF$r8EHWVAL$A-NH2 1453 1518.86 760.43 760.81 734 Ac-ITF$r8EHWAAL$S-NH2 1454 1506.82 754.41 754.8 735 Pam-LTF$r8EYWAQL$S-NH2 1455 1786.07 894.04 894.48 736 Pam-ETF$r8EYWAQL$S-NH2 1456 1802.03 902.02 902.34 737 Ac-LTF$r8AYWLQL$S-NH2 1457 1573.89 787.95 787.39 738 Ac-LTF$r8EYWLQL$S-NH2 1458 1631.90 816.95 817.33 739 Ac-LTF$r8EHWLQL$S-NH2 1459 1605.89 803.95 804.29 740 Ac-LTF$r8VYWAQL$S-NH2 1460 1559.88 780.94 781.34 741 Ac-LTF$r8AYWSQL$S-NH2 1461 1547.84 774.92 775.33 742 Ac-ETF$r8AYWAQL$S-NH2 1462 1547.80 774.90 775.7 743 Ac-LTF$r8EYWAQL$S-NH2 1463 1589.85 795.93 796.33 744 Ac-LTF$r8HYWAQL$S-NHAm 1464 1667.94 834.97 835.37 745 Ac-LTF$r8HYWAQL$S-NHiAm 1465 1667.94 834.97 835.27 746 Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1466 1715.94 858.97 859.42 747 Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1467 1681.96 841.98 842.67 748 Ac-LTF$r8HYWAQL$S-NHnBu 1468 1653.93 827.97 828.24 749 Ac-LTF$r8HYWAQL$S-NHnPr 1469 1639.91 820.96 821.31 750 Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1470 1707.98 854.99 855.35 751 Ac-LTF$r8HYWAQL$S-NHHex 1471 1681.96 841.98 842.4 752 Ac-LTF$r8AYWAQL$S-NHmdPeg2 1472 1633.91 817.96 855.35 753 Ac-LTF$r8AYWAQL$A-NHmdPeg2 1473 1617.92 809.96 810.58 754 Ac-LTF$r5AYWAAL$s8S-NH2 1474 1474.82 738.41 738.79 755 Ac-LTF$r8AYWCouQL$S-NH2 1475 1705.88 853.94 854.61 756 Ac-LTF$r8CouYWAQL$S-NH2 1476 1705.88 853.94 854.7 757 Ac-CouTF$r8AYWAQL$S-NH2 1477 1663.83 832.92 833.33 758 H-LTF$r8AYWAQL$A-NH2 1478 1473.84 737.92 737.29 759 Ac-HHF$r8AYWAQL$S-NH2 1479 1591.83 796.92 797.72 760 Ac-LT2Nal$r8AYWSQL$S-NH2 1480 1597.85 799.93 800.68 761 Ac-LTF$r8HCouWAQL$S-NH2 1481 1679.87 840.94 841.38 762 Ac-LTF$r8AYWCou2QL$S-NH2 1482 1789.94 895.97 896.51 763 Ac-LTF$r8Cou2YWAQL$S-NH2 1483 1789.94 895.97 896.5 764 Ac-Cou2TF$r8AYWAQL$S-NH2 1484 1747.90 874.95 875.42 765 Ac-LTF$r8ACou2WAQL$S-NH2 1485 1697.92 849.96 850.82 766 Dmaac-LTF$r8AYWAQL$S-NH2 1486 1574.89 788.45 788.82 767 Hexac-LTF$r8AYWAQL$S-NH2 1487 1587.91 794.96 795.11 768 Napac-LTF$r8AYWAQL$S-NH2 1488 1657.89 829.95 830.36 769 Pam-LTF$r8AYWAQL$S-NH2 1489 1728.06 865.03 865.45 770 Ac-LT2Nal$r8HYAAQL$S-NH2 1490 1532.84 767.42 767.61 771 Ac-LT2Nal$/r8HYWAQL$/S-NH2 1491 1675.91 838.96 839.1 772 Ac-LT2Nal$r8HYFAQL$S-NH2 1492 1608.87 805.44 805.9 773 Ac-LT2Nal$r8HWAAQL$S-NH2 1493 1555.86 778.93 779.08 774 Ac-LT2Nal$r8HYAWQL$S-NH2 1494 1647.88 824.94 825.04 775 Ac-LT2Nal$r8HYAAQW$S-NH2 1495 1605.83 803.92 804.05 776 Ac-LTW$r8HYWAQL$S-NH2 1496 1636.88 819.44 819.95 777 Ac-LT1Nal$r8HYWAQL$S-NH2 1497 1647.88 824.94 825.41

In some embodiments, a peptidomimetic macrocycles disclosed herein do not comprise a peptidomimetic macrocycle structure as shown in Table 4b.

Table 4c shows examples of non-crosslinked polypeptides comprising D-amino acids.

TABLE 4c SEQ ID Exact Found Calc Calc Calc SP Sequence NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP765 Ac-tawyanfekllr-NH2 1498 777.46 SP766 Ac-tawyanf4CF3ekllr- 1499 811.41 NH2

Example 2: Safety and/or Tolerability Study Study Objectives

This study is designed to (i) evaluate the safety and/or tolerability of peptide 1, which is peptide of this disclosure, administered by IV infusion once weekly for three consecutive weeks of a 21 or a 28-day cycle, and (ii) determine the DLTs and the MTD or OBD of peptide 1 in patients with advanced liquid cancers expressing WT p53 protein. Peptide 1 is an alpha helical hydrocarbon cross-linked polypeptide macrocycle, with an amino acid sequence less than 20 amino acids long that is derived from the transactivation domain of wild type human P53 protein and that contains a phenylalanine, a tryptophan and a leucine amino acid in the same positions relative to each other as in the transactivation domain of wild type human P53 protein. Peptide 1 has a single cross link spanning amino acids in the i to the i+7 position of the amino acid sequence and has more than three amino acids between the i+7 position and the carboxyl terminus. Peptide 1 binds to human MDM2 and MDM4 and has an observed mass of 950-975 m/e as measured by electrospray ionization-mass spectrometry.

Investigational Plan Study Design

This study comprises a dose-escalation, 2-arm study designed to evaluate the safety, tolerability, PK, PD, and anti-liquid cancer cell effects of peptide 1 administered by IV infusion using 2 different dosing regimens of a 28- or 21-day cycle, in patients with advanced liquid cancer (e.g. leukemia, myeloma, and liquid lymphoma) expressing WT p53 protein (see p53 Status Determination below). For example, peptide 1 can be used in patients with relapsed/refractory acute myeloid leukemia (AML) and/or acute lymphoid leukemia (ALL). Patients receive peptide 1 either once weekly for three consecutive weeks for a 28-day cycle or twice weekly for two consecutive weeks for a 21-day cycle. Many patients with a liquid lymphoma present circulating tumor cells (CTC) in peripheral blood, which can be detected and analyzed using flow cytometry. Thus, detection of study drug-specific target engagement in these cells is possible.

The study consists of a Dose Escalation Phase (DEP) and an Expansion Phase (EXP). The DEP is a “3+3” dose escalation design to establish the MTD or OBD of peptide-1. The EXP enrolls up to 2 distinct groups of patients with specific liquid cancers at the MTD or OBD to further investigate the clinical safety profile and potential efficacy of the dose level. The selection of patients for the EXP is finalized based on results of the DEP, as well as data from additional nonclinical pharmacology studies. The later includes the investigation of multiple liquid cancer cell lines (e.g., leukemia, liquid lymphoma, myeloma) that facilitate the comparison of cell line sensitivity to peptide-1 across and within liquid cancer types.

Treatment of patients in the dose escalation and the dose expansion phases of the study continues until documentation of disease progression, unacceptable toxicity, or patient or physician decision to discontinue therapy.

p53 Status Determination and Tumor Sampling Requirement Prior to Enrollment:

A central laboratory tests both archived tissue samples or fresh biopsy samples from all patients enrolled in the study for p53 status using Next-Generation Sequencing (NGS).

For the First 3 Dose Levels of Stage 1:

Patients can be enrolled irrespective of p53 status. Nevertheless, patients are still tested for p53 status at the central laboratory. To this end, archived tissue can be used (e.g., sample must not be older than 3 years), or alternatively, a fresh biopsy can be considered, unless the biopsy poses a significant risk to the patient.

Starting at Dose Level 4 of Stage 1 (and for Patients Enrolled in Stage 2 of the DEP):

Only patients with liquid cancer cells expressing WT p53 protein are enrolled. This key inclusion criterion is based on the proposed mechanism of action of peptide 1, which requires WT p53 protein to be pharmacologically active. The inclusion criterion is also supported by results of in vitro liquid cancer cell growth assays, in which peptide 1 demonstrates activity in liquid cancer cells expressing WT p53 protein, but not in cells with mutations of p53. Patients can meet the p53 requirement through one of the following scenarios:

-   -   Patients can be eligible based on a previous p53 gene test         result done at a local lab. These patients are still tested for         p53 status using NGS at the central laboratory. To this end,         archived tissue can be used (sample must not be older than 3         years), or alternatively, a fresh biopsy should be considered,         unless the biopsy poses a significant risk to the patient.         Patients who do not have archived tissue and for whom a biopsy         poses a significant risk are not enrolled.     -   Patients can be eligible based on archived tissue tested for p53         (sample must not be older than 3 years) at the central lab, or         alternatively, a fresh biopsy can be considered, unless the         biopsy poses a significant risk to the patient. Patients who do         not have archived tissue and for whom a biopsy poses a         significant risk, are not enrolled.

For patients enrolling into the EXP:

-   -   Only patients with liquid cancer cells expressing p53 WT are         enrolled, and all patients must be tested for p53 status using         NGS at the central laboratory PRIOR to enrollment. Archived         tissue can be used (sample must not be older than 1 year), or         alternatively, a fresh biopsy can be considered, unless the         biopsy poses a significant risk to the patient. Patients who do         not have archived tissue and for whom a biopsy poses a         significant risk, are not enrolled.

Only patients with liquid cancer cells expressing WT p53 protein are enrolled. The determination of p53 status is performed on liquid cancer cell samples obtained during the screening period. The assay can be performed by study sites with required capabilities; otherwise it can be performed at a central laboratory. Results from archival tissue samples, if available, can be used to determine patient eligibility in the DEP. The total number of patients enrolled in the study depends on the number of dose levels and the number of patients in each cohort before MTD or OBD is established. Approximately 45 patients, exclusive of replacements for patients who discontinue for non-safety reasons, are enrolled in the DEP, and a total of up to approximately 60 additional patients for each of the up to two patient groups (total of 60) are enrolled in the EXP cohorts. Enrollment of a total of up to 100 patients is planned for the study. Approximately 6 clinical sites in the US are planned. The expected accrual phase is approximately 15 months. The expected follow-up phase is approximately 8 months after the last patient is enrolled, for a total study duration of approximately 23 months.

Patients who satisfy all inclusion and exclusion criteria, including documentation of WT p53 status, are enrolled in cohorts of 3 to 6 patients to receive peptide 1. Peptide 1 is administered by IV infusion in Dose Regimen A over 1 hour (+5 min) on Days 1, 8 and 15 of a 28-day cycle or in Dose Regimen B also over 1 hour (+5 min), starting at Dose Level 3, on Days 1, 4, 8, 11 of a 21-day cycle.

Treatment continues until disease progression, unacceptable toxicity or patient or physician withdrawal of consent. After the MTD or OBD is established, additional patients can be enrolled in up to two separate expansion cohorts (approximately 30 patients per expansion cohort to gain further experience at this dose level and in particular patient or liquid cancer cell types. Selection of patient or liquid cancer cell types is determined in part on the basis of observations made in the dose escalation portion of the study.

Safety is evaluated based on the incidence, severity, duration, causality, seriousness, and type of AE, and changes in the patient's physical examination, vital signs and clinical laboratory results. Investigators use the NCI CTCAE version 4.0 to assess the severity of AEs.

Because the primary objectives of this study are based on safety and PK, statistical analyses are descriptive in nature and accounts for all doses studied and all observed responses, including patients who achieve a complete response (CR) or partial response (PR) or who maintain stable disease (SD) based on IWG (2014) criteria. Patients who receive at least one dose of peptide 1 constitute the safety population and are included in all safety analyses. Patients who complete at least one cycle of peptide 1 and undergo a post-treatment objective disease assessment constitute the efficacy-evaluable patient population.

Patient Population Inclusion Criteria

All AML patients are required to have relapsed or refractory acute myeloid leukemia according to WHO criteria. Patients with acute promyelocytic leukemia are excluded. All MDS patients are required to have: (i) Diagnosis of MDS confirmed within 8 weeks prior to study entry; (ii) Not responsive to or intolerant to hypomethylating agents (azacytidine or decitabine); (iii) IPSS-R intermediate/high/very high-risk MDS patients (applying IPSS-R criteria at screening); or, if IPSS-R status cannot be determined (e.g., if cytogenetics are not available due to dry tap), FAB classification: RAEB-1 (5% to 9% BM blasts), RAEB-2 (10% to 19% BM blasts), CMML (10% to 20% BM blasts) and WBC<13,000/LL, RAEB-t (20% to 30% BM blasts); (iv) MDS patients must also meet at least one of the following: a. Progression (according to 2006 IWG criteria) at any time after initiation of azacitidine or decitabine treatment; b. Failure to achieve complete or partial response or hematological improvement (according to 2006 IWG) after at least six 4-week cycles of azacitidine or decitabine; c. Relapse after initial complete or partial response or hematological improvement (according to 2006 IWG criteria) observed after at least four 4-week cycles of azacitidine or decitabine; d. Intolerance to azacitidine or decitabine defined by drug-related ≥Grade 3 liver or renal toxicity leading to treatment discontinuation.

All patients are required to meet the following inclusion criterias: (i) Male or female patients age 18 years and older, inclusive, at the time of informed consent (ii) Histologically- or cytologically-confirmed liquid cancers. Standard curative measures do not exist or are no longer effective; (iii) WT p53 status for the relapsing or treatment-refractory liquid neoplasm is mandatory for patients enrolling at Dose Level 4 and higher in Stage 1 of the DEP, as well as for all patients enrolled in Stage 2 of the DEP or in the EXP; (iv) at least one target lesion that is measurable by Revised International Working Group Response Criteria (IWG (2014)) in liquid lymphoma patients (v) ECOG performance status 0-1; (vi) predicted life expectancy of ≥3 months; (vii) adequate hematologic function, measured within 7 days prior to the first dose of peptide 1 (defined as: ANC≥1.5×10⁹/L, Hemoglobin ≥9.0 g/d, and Platelets ≥100×10⁹/L); (viii) adequate hepatic function, measured within 7 days prior to the first dose of peptide 1 (defined as: in the absence of disease involvement in the liver:bilirubin ≤1.5 times institutional ULN: AST and ALT≤2.5 times ULN; in the presence of disease involvement in the liver:bilirubin ≤2 times ULN: AST and ALT≤5 times ULN, (ix) adequate renal function, measured within 7 days prior to the first dose of peptide 1, (defined as: urinalysis with no evidence of +2 or higher proteinuria, serum creatinine ≤1.5 times institutional ULN or calculated creatinine clearance ≥50 mL/min (Cockcroft-Gault formula)); (x) acceptable coagulation profile, measured within 7 days prior to the first dose of peptide 1 (defined as: PT or INR≤1.5 times ULN; aPTT≤1.5 times ULN); (Xi) at least 4 weeks since prior chemotherapy or biologic therapy, radiotherapy or surgery (intra-thoracic, intra-abdominal or intra-pelvic) with recovery to Grade 1 or baseline of significant toxicities, excluding alopecia, from previous therapies. Palliative radiotherapy for bone lesions ≤2 weeks prior to the first dose of peptide 1 is acceptable if acute toxicity has resolved; (xii) negative serum pregnancy test within 14 days prior to the first dose of peptide 1 for women of child-bearing potential, defined as a sexually mature woman who has not undergone a hysterectomy or who has not been naturally postmenopausal for ≥24 consecutive months (i.e., who has had menses any time in the preceding 24 consecutive months); (xiii) all patients (males and females) of child-bearing potential agree to use an effective method of birth control (i.e., latex condom, diaphragm, cervical cap, IUD, birth control pill, etc.) beginning two weeks before the first dose of peptide 1 and for 30 days after the last dose of peptide 1; (xiv) ability to understand and willingness to sign a written informed consent document; and patients with prostate cancer must continue androgen deprivation therapy, unless such therapy was discontinued 6 months prior to first dose of peptide 1. In a study of using peptide 1 for acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL), patients with pathological confirmation of AML or ALL, such as B-cell acute lymphoid leukemia (B-ALL) and T-cell acute lymphocytic leukemia (T-ALL), are included. In such study, patients who are relapsed, refractory or intolerant to standard chemotherapy are included.

Exclusion Criterias

Patients who meet any of the following criteria at screening or Day-1 are excluded: (i) previous treatment with investigational agents that affect MDM2 or MDMX activity; known hypersensitivity to any study drug component; (iii) known and untreated brain metastases. Patients with brain metastases that have been treated and demonstrated to be clinically stable for ≥30 days can be enrolled onto the dose escalation portion of the study; (iv) history of coagulopathy, platelet disorder or history of non-drug induced thrombocytopenia; (v) history of pulmonary embolism within 6 months prior to the first dose of peptide 1 or untreated DVT; (vi) required concurrent use of anti-coagulants or anti-platelet medication, with the exception of aspirin doses ≤81 mg/day, low-dose SC heparin or SC low-molecular-weight heparin for DVT prophylaxis, or heparin flushes to maintain IV catheter patency; (vii) patients with pre-existing history of or known cardiovascular risk (for example: history of acute coronary syndromes including myocardial infarction, unstable angina, coronary artery bypass graft, angioplasty, or stenting within 6 months prior to the first dose of peptide 1; uncontrolled hypertension defined as a systolic BP≥160 mmHg and/or diastolic BP≥100 mmHg; pre-existing cardiac failure (New York Heart Association class III-IV); atrial fibrillation on anti-coagulants; clinically significant uncontrolled arrhythmias or arrhythmia requiring treatment, with the exceptions of atrial fibrillation and paroxysmal supraventricular tachycardia; severe valvulopathy; corrected QTc interval on screening ECG≥450 msec for males and ≥470 msec for females); (viii) clinically significant gastrointestinal bleeding within 6 months prior to the first dose of peptide 1; (ix) clinically significant third-space fluid accumulation (e.g., ascites requiring tapping despite the use of diuretics, or pleural effusion that requires tapping or is associated with shortness of breath); (x) pregnant or lactating females; (xi) evidence of serious and/or unstable pre-existing medical, psychiatric or other condition (including laboratory abnormalities) that could interfere with patient safety or provision of informed consent to participate in this study; (xii) active uncontrolled infection, a history of HIV/AIDS, or a history of hepatitis B or C in the absence of hepatocellular carcinoma. Patients with primary liver cancer that have positive hepatitis serology but are not demonstrating active viral hepatitis can be considered for enrollment if they meet all other inclusion and no other exclusion criteria; (xiii) starting at dose level 4 and higher in Stage 1 of the DEP (as well as for all patients enrolling in Stage 2 of the DEP or in the EXP): Cancers with known Human Papilloma Virus (HPV)-association such as HPV-positive cervical cancers, HPV-positive oropharyngeal cancers or HPV-positive anal cancers; (xiv) known history of another primary malignancy that has not been in remission for ≥2 years. Non-melanoma skin cancer and cervical carcinoma in situ or squamous intraepithelial lesions (e.g., CIN or PIN) are allowed; (xv) any psychological, sociological, or geographical condition that could potentially interfere with compliance with the study protocol and follow-up schedule; (xvi) the required use of any concomitant medications that are predominantly cleared by hepatobiliary transporters (e.g., OATP members OATP1B1 and OATP1B3) within 24 hours of peptide 1 infusion; (xvii) the use of any investigational agents within 4 weeks or 5 circulating half-lives prior to the first dose of peptide 1. In a study of using peptide 1 for acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL), patients with acute undifferentiated or biphenotypic leukemia are excluded. Patients with a leukemic blast count of more than 50,000/uL are excluded. Patients with deletion of chromosome 17, or del(17p) are excluded.

Patient Removal/Replacement from Study Therapy

A patient can be removed from the study therapy for a variety of reasons, including: (i) disease progression; (ii) unacceptable adverse event(s); (iii) intercurrent illness that prevents further participation; (iv) clinically significant toxicity despite a 2-week dosing delay or after two dose reductions; (v) patient refusal to continue treatment through the study and/or consent withdrawal for study participation; (vi) patient unable or unwilling to comply with study requirements; (vii) pregnancy or failure to use adequate birth control; (viii) general or specific changes in the patient's condition that render the patient unacceptable for further treatment in this study in the judgment of the investigator

Any patient who completes screening and does not receive a dose of peptide 1 is replaced. A patient in the dose escalation portion of the study who discontinues the study prior to completion of the first cycle for reasons other than safety is replaced. A patient in the dose expansion portion of the study who discontinues study participation prior to the completion of the first cycle of treatment for any reason can be replaced.

Treatment Plan Study Drug Administration

The study drug is the investigational agent peptide 1. This investigational agent is distributed to clinical sites. Patients begin treatment with peptide 1 within 21 days following the start of screening. Peptide 1 drug is a frozen liquid product supplied in single-use glass vials. The peptidomimetic macrocycle for injection is stored frozen at ≤−15° C. Peptide 1 is introduced into an IV infusion bag containing D5W; this is known as peptide 1 dosing solution and is provided by the site pharmacy for administration to the patient. Peptide 1 dosing solution is labeled with a patient identification number. An investigative staff confirms this information and its relevancy to the intended patient. Peptide 1 is administered by IV infusion in D5W over 1 hour (+5 min) on Days 1, 8 and 15 of a 28-day cycle for DR-A or Days 1, 4, 8, 11 for DR-B of a 21 day treatment cycle. The pre-defined dose is calculated for each patient based on body weight at the start of each cycle.

Peptide 1 is not administered outside of the planned schedule for Dose Regimens A and B in Cycle 1, i.e., there are no planned windows for dose days. Follow-up visits on non-dosing days have a window of +1 day in DR-A on Days 22 and 29 and in DR-B on Days 18 and 21. Deviations are noted on the eCRF. Treatment of patients in the dose escalation and the dose expansion phases of the study continue until documentation of disease progression, unacceptable toxicity, or patient or physician decision to discontinue therapy.

In case of infusion-related reactions, peptide 1 infusion is temporarily discontinued. Pharmacologic agents and other therapeutic interventions can be administered per institutional guidelines. The decision to re-start peptide 1 infusion is made after a careful assessment of the patient.

Starting Dose, Dose Escalation and Dose Reduction

Starting at Dose Level (DL) 3, patients are sequentially assigned to one of two treatment arms: Dose Regimen (DR) A continues testing administration of peptide 1 once per week, or Dose Regimen (DR) B testing administration of peptide 1 twice per week. For Dose Level 3, DR-A is enrolled first, DR-B is enrolled second. The starting dose (DL-1) in DEP, based on results from nonclinical toxicology assessments, is 0.16 mg/kg. During the first 2 dose levels, patients receive peptide 1 on Days 1, 8, and 15 of a 28-day cycle. Starting with DL 3, patients in DR-A continues being treated once a week on Days 1, 8, and 15 of a 28-day cycle, whereas patients in DR-B are treated twice a week, on Days 1 and 4, 8 and 11 of a 21-day cycle.

Dose Levels for the Dose Escalation Portion of Study

In the Dose Escalation portion of the study, increasing dose levels of peptide 1 are evaluated in cohorts of 3-6 patients. Peptide 1 is administered by IV infusion using 2 different dosing regimens of a 28- or 21-day cycle, in patients with advanced liquid lymphomas expressing WT p53 protein. Patients receive peptide 1 either once weekly for three consecutive weeks for a 28-day cycle or twice weekly for two consecutive weeks for a 21-day cycle. Many patients with a liquid lymphoma present circulating tumor cells (CTC) in peripheral blood, which can be detected and analyzed using flow cytometry. This analysis allows detection of study drug-specific target engagement in these cells. Based on allometric scaling, the projected AUC in humans at 0.16 mg/kg (50 μg·hr/mL) is approximately 9% of the rat AUC at STD₁₀ and approximately 6% of the AUC at the monkey HNSTD.

In the absence of DLT in ≥33% of patients in either DR, subsequent cohorts of 3 to 6 patients will receive escalated doses until the MTD or an OBD is established for each dose regimen.

A 2-stage dose escalation design is employed. During the initial Stage 1 Escalation Phase (Table 5), 100% dose increments are utilized until ≥1 of 3 patients in a cohort experiences any Grade ≥2 AE that is at least possibly related to study drug. Subsequent dose escalation will continue using 3-patient cohorts and the modified Fibonacci sequence (i.e., Stage 2 Escalation Phase; Table 6), until the MTD or OBD is established.

TABLE 5 Dose Level and Dose Regimen Schematic Dose Escalation Phase (DES) Dose Expansion Dose Dose Dose Dose Dose Dose Dose Phase Level Level Level Level Level Level Level Dose(s) & 1 2 3A 4A 5A 6A 7A Regimen(s) Per Dose, mg/Kg to be 0.16 0.32 0.64 1.25 2.5  5.0 10.0 Determined mg/Kg mg/Kg mg/Kg mg/Kg mg/Kg mg/Kg mg/Kg P53 WT Pre-Dose 1 Only Dose Regimen A DR-A: 1x/wk for 3 wks, 28 day Cycle Fresh Sample Liquid Cancer Only DR-B: 2x/wk for 2 wks, 21 Day Cycle 0.32 0.64 1.25 2.5  5.0 mg/Kg mg/Kg mg/Kg mg/Kg mg/Kg Per Dose, mg/Kg Dose Dose Dose Dose Dose Level Level Level Level Level 3B 4B 5B 6B 7B ←p53 WT Not Necessary→ ←p53 WT Pre-Dose 1 Necessary→ Archive Sample Used if Available From Archive or Fresh Sample Dosing Overview Begin Next Dose Regimen A Clinic Visits-28 Day Cycle Cycle ClinicalScreen Dose: Day 2 Day 3 Dose: Dose: Day 16 Day 22 Day 29 Day-21 Day 1 Day 8 Day 15 to Day-1 Dose Regimen B Clinic Visits-21 Day Cycle Begin Next Cycle Dose: Day 2 Day 3 Dose: Dose: Dose: Day 12 Day 18 Day 22 Day 1 Day 4 Day 8 Day 11 Peptide 1 Administration for each Dose Level (DL) and Dose Regimen Dose Dose-Day 1 Dose-Day 8 Dose-Day 15 28-Day Total Dose Regimen A mg/Kg mg/Kg mg/Kg Cycle per Cycle mg/Kg Dose Level 3 0.64 0.64 0.64 DL 3-1.92 DL 4 1.25 1.25 1.25 DL 4-3.75 DL 5 2.5 2.5 2.5 DL 5-7.5  DL 6 5.0 5.0 5.0 DL 6-15.0 DL 7 10.0 10.0 10.0 DL 7-30.0 Dose Dose-Day 1 Dose-Day 4 Dose-Day 8 Dose-Day 11 21-Day Total Dose Regimen B mg/Kg mg/Kg mg/Kg mg/Kg Cycle per Cycle mg/Kg Dose Level 3 0.32 0.32 0.32 0.32 DL 3-1.28 DL 4 0.64 0.64 0.64 0.64 DL 4-2.56 DL 5 1.25 1.25 1.25 1.25 DL 5-5.0  DL 6 2.5 2.5 2.5 2.5 DL 6-10.0 DL 7 5.0 5.0 5.0 5.0 DL 7-20.0

The escalation scheme can be switched to the Stage 2 Escalation Schedule at any point that the Investigators, Sponsor's Medical Monitor and Safety Physician representative agree on a more conservative progression.

The observation of DLT(s) is used to make individual patient determinations regarding dose reductions, interruptions or discontinuation throughout the course of the trial, but DLTs occurring during Cycle 1 is used to inform safety and tolerability assessments for dose escalation decisions.

If DLTs are observed in the first cohort, the dose is de-escalated to Dose Level-1. If DLTs are observed at Dose Level-1, the dose is de-escalated to Dose Level-2. If DLTs are observed at Dose Level-2, other dose levels can be considered and implemented after discussions among the Investigators, Sponsor's Medical Monitor and Safety Physician representative.

Within each Dose Regimen:

If no DLT is observed in a cohort, the subsequent patient group is enrolled at the next planned dose level of that dose regimen.

If DLT is observed in ≥2 of 3 patients at any dose level no further dose escalation occurs in that DR, and the current dose is defined as the MAD.

If DLT is observed in 1 of 3 patients at any dose level, then up to 3 additional patients are enrolled in the same DR at that same dose level. If DLT is observed in ≥2 patients in the expanded cohort, then no further dose escalation occurs, and the current dose is defined as the MAD.

After the MAD is defined, either the previously administered lower dose is expanded to a total of 6 patients, or an intermediate (between the MAD and the next lower dose level) is investigated in up to six patients. The highest dose tolerated without DLT in at least 5 of 6 patients in a cohort from one treatment arm is defined as the MTD or OBD for that treatment arm.

The selection of dose regimen and dose level for up to 2 EXP cohorts is based on the MTD determination in Cycle 1, as well as the cumulative safety, efficacy and PK/PD profile of peptide 1 in subsequent treatment cycles in DEP.

Dose levels are be increased between cycles within each cohort, and patients are assigned only one dose level (i.e., no intra-patient dose escalation).

Dose Level for the Expansion Portion of Study

After the MTD or OBD is defined, approximately 30 additional patients can be enrolled in up to two expansion cohorts of the study to gain further experience at this dose level and investigate the effect of peptide 1 in specific patient or liquid cancer cell types. There can be up to two expansion cohorts, for which two disease types can be selected for evaluation. The dose and dosing schedule of peptide 1 administered to patients in the expansion cohort is derived from evaluation of available safety and other information from patients from both dose regimens in the dose escalation portion of the study.

Intra-Patient Dose Escalation

Intra-patient dose escalation is not be permitted.

Dose and Schedule Adjustments for Toxicity

Toxicity that occurs during a cycle must recover as outlined below for treatment to continue. Hemoglobin ≥8.5 g/dL; ANC≥1.0 10⁹/L; platelet count ≥75×10⁹/L; liver function tests back to grade prior to previous cycle (includes PT/INR); other toxicities must return to Grade ≤1 or to baseline level if Grade ≥1 was acceptable for inclusion in the trial as described in the criteria listed in Section 4.

In the event a Grade 4 AE considered related to peptide 1 is observed, peptide 1 must be discontinued permanently. Exceptions include Grade 4 neutropenia lasting <3 days, and emesis, diarrhea or electrolyte abnormalities that resolve within 2 days on optimum treatment. For these exceptions, treatment can be delayed for up to 2 weeks to allow resolution of the toxicity (i.e., return to Grade ≤1 or baseline), followed by re-treatment at a reduced dose. Two dose reductions are permitted. A third dose reduction requires evidence of clinical benefit and approval by the Medical Monitor.

In the event a Grade 3 AE considered related to peptide 1 is observed (exceptions are Grade 3 nausea, emesis, diarrhea or clinically insignificant electrolyte abnormalities that resolve within 2 days on optimum treatment), treatment can be delayed for up to 2 weeks to allow resolution of the toxicity, followed by re-treatment at a reduced dose. Two dose reductions are permitted. A third dose reduction require evidence of clinical benefit and approval by the Medical Monitor.

Dose modifications for re-treatment following related Grade 3 and Grade 4 AEs (as permitted) is as follows. For DEP, patients are re-treated at the preceding dose level (per Table 1 and/or Table 2). For EXP, doses are reduced by 25% intervals (e.g., if the Phase II dose is 5 mg/kg, the dose is reduced sequentially to 3.75 mg/kg and 2.8 mg/kg). Two dose reductions are permitted. A third dose reduction requires evidence of clinical benefit and approval by the Medical Monitor.

If a clinically significant AE is observed in a patient during a treatment cycle, further dosing is delayed until the toxicity has resolved to an acceptable level. Treatment can be delayed by up to 2 weeks to allow for the resolution of AEs, and a dose reduction to the preceding level can be made at the discretion of the Investigator in consultation with Sponsor's Medical Monitor and Safety Physician representative. If a patient experiences multiple AEs, decisions on dosing delay or dose reduction can be based on the most severe AE. Any patient who experiences recurrent, clinically significant AE after one dose reduction can undergo one additional dose reduction. Patients who continue to experience clinically significant toxicity after a 2-week delay or the maximum allowed number of dose reductions are discontinued from the study.

Adverse events considered for dose reduction do not include the events assessed by the investigator as exclusively related to underlying disease or other medical condition or concomitant treatment. A patient who experiences an AE considered related to peptide 1 can continue on study if the patient is receiving clinical benefit and/or the Investigator feels continued participation is in the best interest of the patient. In such cases, at the Investigator's discretion and in agreement with Sponsor's Medical Monitor and Safety Physician representative, the dose for a patient can be reduced to the preceding lower level.

Up to two dose reductions for a patient are permitted. A third dose reduction requires evidence of clinical benefit and approval by the Medical Monitor, after which the patient is discontinued from the study.

A patient who experiences a DLT can continue treatment at the preceding lower level at the discretion of the Investigator and in agreement with Sponsor's Medical Monitor and Safety Physician representative until disease progression or unacceptable toxicity. Once the dose has been reduced for a patient, it can not be re-escalated.

Toxicity grading is based on NCI CTCAE v4.0.

Statistical Methods

Statistical analyses of safety and efficacy for DEP and EXP are primarily descriptive in nature because the objectives of the study are to determine the DLTs and MTD or OBD. These objectives are achieved by the results of a deterministic algorithm: Continuous variables are summarized using descriptive statistics [n, mean, standard deviation, median, minimum, and maximum]. Categorical variables are summarized showing the number and percentage (n, %) of patients within each classification. Results are evaluated for all patients and liquid lymphoma patients. Results from DR-A and DR-B are compared for all Dose Levels and patient groups.

Example 3: Study Procedures Schedule of Study Events

The schedule of study activities, including assessments, tests, exams, disease assessments, submission of tissue specimens, and study drug administration) that is conducted, beginning with screening and continuing through Cycle 1 [day 1, day 8, and day 15 of a 28 day cycle] is outlined in Table 7. The study that is conducted beginning with Cycle 2 [day 29 of cycle 1=day 1 of cycle 2] is outlined in Table 8.

TABLE 7 Schedule of study activities through Cycle 1 Day 22 (DR- B only) Day 1, Cycle 2 Day 29/ Day Day Day Clinical 12 18 1, Screen- (DR- (DR- Cycle 21 Day- Day 2 ± Day 3 ± B B 2 ± days 7 Day 1 4 4 Day 8 Day 11 only) only) 3 d for for for DR- hfor hfor for DR- (DR-A Day Day (DR- DR-A DR-A A and DR-A DR-A A and only) 16 22 A and and DR-B, and and Day 4 DR-B, Day 15 (DR- (DR- only) DR-B, DR-B, All Dose DR-B, DR-B, (DR-B All Dose (DR-B A A Refer All All Levels All All only) Levels only) only) only) to Molecular Dose Dose pre- post- Dose Dose pre- post- pre- post- pre- post- ± ± Table Screen Levels Levels dose dose Levels Levels dose dose dose dose dose dose 2 d 1 d 7 Written X X informed consent Medical history X Demographics X Tumor biopsy X X or archive tissue sample for p53 WT confirmation and biomarker assessment Confirm X X eligibility Blood test for X HIV, hepatitis B and C Serum or urine X pregnancy Vital signs: X X X X X X X X X X X X X Blood pressure, pulse, respiration rate, body temperature Physical exam X X X X 12-lead ECG X X X X X Laboratory X X X X X X X X X assessments: Clinical chemistry (glucose, calcium, albumin, total protein, sodium, potassium, CO₂, chloride, BUN [blood urea nitrogen], serum creatinine, uric acid, ALP, ALT, AST, phosphate, total and direct bilirubin), hematology (complete blood count, platelets and differential), urinalysis (dipstick measurement [pH, specific gravity, protein, glucose, ketones, nitrite, leukocyte esterase] with microscopic analysis, if results of the dipstick indicate additional testing required), coagulation (PT, INR, aPTT). Collection of X X blood for immunogenicity Collection of X X X X X X X X X X X blood for biomarker assessments Collection of X X X X X X X X X X blood for PK assessments Collection of X blood for PK assessments FLT-PET for X patients who received FLT- PET at screen and have SUV ≥5 ECOG X X X Performance Status Needle biopsy X X for biomarker assessments Tumor X Assessment peptide 1 X X dosing Concomitant X X X X X X X X X X X X X medications AE assessment X X X X X X X X X X TLS monitoring X (via routine laboratory assessment sample)

TABLE 8 Dose Regimen A-Study Activities Through Cycle 1 Within 7 days Day 29/ prior Day Day Day 1, Clinical to Day 1 2 ± 3 ± Day 8 ± 1 d Day 15 ± 1 d Day Day Cycle Molecular Screen-21 Day Pre- Post- 4 4 Pre- Post- Pre- Post- 16 ± 2 22 ± 1 2¹ ± (DR-A) Screen days 1 Dose Dose h h Dose Dose Dose Dose d d 3 d Written X X informed consent Medical X history² Demographics X X Pre-dose CT & FDG-PET/ FLT possibly Tumor X biopsy or archive tissue sample for p53 WT confirmation DL 4 and beyond and biomarker assessment Confirm X X eligibility Blood test X for HIV, hepatitis B and C Serum or X urine pregnancy Vital signs X X X X X X X X X X X X Physical X X X X exam 12-lead ECG X X X Laboratory X X X X X X X X assessments Blood X Collection- immunogenicity Blood X X X X X X X X X X Collection- biomarker assessments Blood X X X X X X X X X Collection- PK assessments ECOG X X X X Performance Status Needle X biopsy for biomarker assessments Tumor X Assessment peptide 1 X X X dosing Concomitant X X X X X X X X X X X X medications AE X X X X X X X X X assessment

TABLE 9 Dose Regimen B-Study Activities Through Cycle 1 Within 7 days Day 21/ prior Day Day Day 1, Clinical to Day 1 2 ± 3 ± Days 4 and 8 Day 11 Day Cycle Molecular Screen-21 Day Pre- Post- 4 4 Pre- Post- Pre- Post- Day 18 ± 1 2¹ ± (DR-B) Screen days 1 Dose Dose h h Dose Dose Dose Dose 12 d 2 d Written X* X** informed consent Medical X history Demographics X X Pre-dose CT & FDG-PET/ FLT possibly Tumor biopsy X or archive tissue sample for p53 WT confirmation DL 4 and beyond, and biomarker assessment Confirm X X eligibility Blood test for X HIV, hepatitis B and C Serum or urine X pregnancy Vital signs X X X X X X X X X X X X Physical exam X X X X 12-lead ECG X X X Laboratory X X X X X X X X assessments Blood X X Collection- immunogenicity Blood X X X X X X X X X X Collection- biomarker assessments Blood X X X X X X X X X Collection- PK assessments ECOG X X X X Performance Status Needle biopsy X for biomarker assessments Tumor X Assessment peptide 1 X X X dosing Concomitant X X X X X X X X X X X X medications AE assessment X X X X X X X X X

TABLE 10 Schedule of study activities through Cycle 2 Day 29 for DR-A and Day 22 for DR-B of prior cycle/Day 1 of next cycle for Day 15 of Day 16 patients Day 8 of DR-A DR-A and DR-A End-of- continuing and Days 4 and Day 11 of and Study treatment ± 8 of DR-B ± DR-B ± Day 12 CT 30 ± 3 d after 3 d 1 d 1 d of DR- After even Imaging* last dose or Pre- Post- Pre- Post- Pre- Post- B ± numbered Prior to study Dose Dose Dose Dose Dose Dose 2 d cycles first dose withdrawal Serum X pregnancy Vital signs: X X X X X X X X Blood pressure, pulse, respiration rate, body temperature. Physical exam X X X X 12-lead ECG X X At X pre-dose pre-dose and EOI and EOI (+10 (+10 min) min) Laboratory X X X X X assessments: (Hematology Clinical only) chemistry, hematology, urinalysis, coagulation (PT, INR, aPTT). Collection of X X X blood for immunogenicity Collection of X X X X X X blood (MIC-1 (MIC-1 (MIC- (MIC- for biomarker only) only) 1 1 assessments only) only) (each cycle) Collection of X X X X X X blood for PK assessments (Cycle 2 and End-of-Study only) ECOG X X X X Performance status Needle Biopsy X for biomarker assessments Tumor X X assessment At end of even- numbered cycles. Prior to start of the next treatment cycle peptide 1 X X X dosing Concomitant X X X X X X X X medications FLT-PET provided that an evaluable FDG- PET-scan was performed prior to starting treatment AE assessment X X X X X X X X (begins at the point of the first peptide 1 infusion and continues until 30 days after last infusion) *All patients receive a CT image prior to the first dose. After dosing commences, in DR-A: CT images obtained at the end of Cycle 2 and every other cycle thereafter in DR-A, e.g., Cycles 4, 6, and 8 DR-B: CT images obtained after the last infusion in Cycle 3 and every third cycle thereafter in DR-B, e.g., Cycles 6, 9, and 12. Images are obtained after the last dose is administered in those cycles and prior to the Day 18 visit.

TABLE 11 Dose Regimen A-Study Activities Cycle 2 and Beyond Day 29 of prior cycle/ End-of-Study Day 1 ± Day 8 ± Day 15 ± After 30 ± 2 d after 3 d 1 d 1 d even last dose or Pre- Post- Pre- Post- Pre- Post- Day 16 ± numbered study (DR-A) Dose Dose Dose Dose Dose Dose 2 d cycles wiyhdrawal Serum or urine X pregnancy Vital signs X X X X X X X X Physical exam X X X X 12-lead ECG X X X Laboratory X X X X X assessments Collection of blood X X X for immunogenicity Blood Collection- X X X X X X biomarker assessments (each cycle) Blood Collection- X X X X X X PK assessments (Cycle 2 and End-of- Study only) ECOG Performance X X X X status Needle biopsy for X biomarker assessments CT Imaging X X peptide 1 dosing X X X Concomitant X X X X X X X X medications AE assessment X X X X X X X X

Table 12: Dose Regimen B-Study Activities Cycle 2 and Beyond Example 4: Pharmacokinetic Analysis

Levels of peptide 1 and its metabolites are measured in blood samples collected at specific time points described below. Pharmacokinetic data are tabulated and summarized by individual patient and collectively by dose level for each dose regimen. Graphical displays are provided where useful in the interpretation of results.

Blood samples for PK assessment are collected at the following time points:

TABLE 13 Time points for collection of blood samples for PK assessment Cycle 1 Day 1 within one hour before SOI DR-A and DR-B EOI (+5 min) 30 min after EOI (±5 min) 1 hr after EOI (±5 min) 2 hr after EOI (±10 min) 4 hr after EOI (±10 min) 8 hr after EOI (±10 min) Day 2 24 hours (±4 hr) after SOI day prior DR-A and DR-B Day 3 48 hours (±4 hr) after SOI Day 1 DR-A and DR-B Day 8, DR-A within one hour before SOI Days 4 & 8, DR-B EOI (+5 min) 30 min after EOI (±5 min) 1 hr after EOI (±5 min) 2 hr after EOI (±10 min) 4 hr after EOI (±10 min) Day 15, DR-A within one hour before SOI Day 11, DR-B EOI (+5 min) 30 min after EOI (±5 min) 1 hr after EOI (±5 min) 2 hr after EOI (±10 min) 4 hr after EOI (±10 min) 8 hr after EOI (±10 min) Day 16, DR-A 24 hours (±4 hrs) after SOI day prior Day 12, DR-B Cycle 2 Cycle 1 Day 29/ within one hour before SOI Cycle 2 Day 1, DR-A EOI (+5 min) Cycle 1 Day 23/ 30 min after EOI (±5 min) Cycle 2 Day 1, DR-B 1 hr after EOI (±5 min) 2 hr after EOI (±10 min) 4 hr after EOI (±10 min) SOI stands for start of infusion of peptide 1; EOI stands for the end of infusion peptide 1.

Example 5: Pharmacokinetic Study

Pharmacokinetic studies characterize exposure kinetics following single IV administrations of peptide 1 in mice, rats and monkeys, including evaluations of two different dosing formulations in rats and monkeys. Using qualified liquid chromatography with tandem mass spectrometry (LC-MS-MS) methods for efficacy models and dose range-finding (DRF) studies, and validated methods for GLP safety studies, absorption was characterized in mice at the MED in efficacy models and in rats and monkeys at tolerated and non-tolerated doses in toxicology studies. Exposures generally increased proportionally with dose, although an apparent plateau was observed at the highest dose of the 4-week monkey toxicology study. No sex-based differences were observed in either species, and no accumulation was observed following multiple doses.

The in vitro protein binding of peptide 1 was evaluated over a range of concentrations in mouse, rat and monkey plasma, as well as human plasma samples from normal subjects and hypoalbuminemic patients. Protein binding ranged from 92% to 98% in plasma of mice, rats, dogs, monkeys, and humans following incubation of peptide 1 at a single concentration of 2 M, and exceeded 98% in mouse and rat plasma up 250 M. In human and monkey plasma, free peptide 1 fractions of 3-4% were measured at peptide 1 concentrations up to 150 M, corresponding to expected Cmax values from clinical doses up to 15 mg/kg, rising to 12-14% at concentrations >200 μM. In plasma from hypoalbuminemic patients, a similar rise was seen at >100 μM concentrations of peptide 1, corresponding to expected Cmax values from clinical doses up to 10 mg/kg. The concentration-dependent plasma protein binding is consistent with the plateau in exposure observed at the high-dose group (20 mg/kg) in the 4-week monkey GLP toxicity study, and suggests less-than-dose-proportional exposure at very high clinical doses, in particular for patients with hypoalbuminemia.

In vitro studies demonstrated a similar metabolite profile across species, including humans, providing support for the rat and the monkey as suitable species for toxicology studies. Proteolysis is the major biotransformation pathway of peptide 1. The predominant metabolite is a 3-amino acid truncation with the cyclic peptide portion intact, and the same metabolite profile was noted in in vitro stability studies with mouse, rat, monkey, and human cryopreserved hepatocytes. In a single-dose rat study, hepatobiliary metabolism and elimination represented the predominant clearance pathway for peptide 1, with the predominant metabolite being the major excretion product observed in the bile. The predominant metabolite was also observed in the plasma in both the rat and monkey 4-week GLP toxicology studies, with adequate exposures in these studies to provide characterization of its impact on the overall safety profile of peptide 1. In the monkey, the predominant metabolite plasma exposure was 10% of the predominant metabolite AUC, and in the rat, the predominant metabolite exposure was 6% of the peptide 1 AUC. Accumulation of the predominant metabolite was not observed with repeated twice-weekly dosing in rats or monkeys.

Inhibition or induction of cytochrome P450 (CYP) enzymes by peptide 1 appears to be negligible at clinically-relevant concentrations, although interactions are possible at high exposures of peptide 1 with drugs that are predominantly cleared by hepatobiliary transporters.

Example 6: Pharmacodynamic Analysis

Levels of p53, MDM2, MDMX, p21 and caspase are measured in liquid cancer cell specimens collected before beginning treatment and at the end of Cycle 1 or Cycle 2. MIC-1 is measured in blood samples. The specific time points for blood and tissue collection for PD assessments are described below. Pharmacodynamic data are tabulated and summarized by individual patient and collectively by dose level. Graphical displays are provided where useful in the interpretation of results.

Results available from previous genetic and biomarker tests, and additional tests of the blood and liquid cancer cell samples for biomarkers relevant to the safety and efficacy of peptide-1 can be investigated for possible correlation with patient outcome.

Blood samples for PD assessments are collected at the following time points:

TABLE 14 Time points for collection of blood samples for PD assessments Cycle 1 DR-A, DR-B, or Both: Dose Regimens Assessment Blood Sample Collection Schedule Day 1-Both (pre) MIC-1 and within 1 hour before the CTC start of infusion (SOI) Day 1-Both (post) Samples EOI (+5 min) & EOI + 1 hr (±5 min), 2, 4, and 8 hr (+10 min) Day 2- Both 24 hours (±4 hr) after SOI on Day 1 Day 3- Both 48 hours (±4 hr) after SOI on Day 1 Day 8 DR-A within 1 hour before SOI and Day 4 & 8 DR-B within 1 hour after the end of infusion (EOI) Day 15 DR-A within 1 hour before SOI and Day 11 DR-B within 1 hour after EOI Day 15 DR-A within 1 hour before SOI and Day 11 DR-B EOI (+5 min) & EOI + thr (±5 min), 2, 4, and 8 hr (±10 min) Day 16 DR-A 24 hours (±4 hrs) after SOI day prior Day 12 DR-B Day 22 DR-A During Day visit Day 18 DR-B Each Subsequent Cycle Starting in Cycle (Cy) 2 Dose Regimens Assessments Blood Sample Collection Schedule Cy 1 Day 29 DR-A MIC-1 within 1 hour before SOI and Cy 1 Day 23 DR-B = Only within 1 hour after EOI Cycle 2 Day 1 Day 15 DR-A within 1 hour before SOI and Day 11 DR-B within 1 hour after EOI Day 16 DR-A 24 hours (±4 hrs) after SOI day prior Day 12 DR-B End of study visit During end of study visit NOTE: no PD assessments for liquid lymphoma on Day 8 DR-A or Days 4 and 8 DR-B

Example 7: Assessment of Clinical Activity of the Peptidomimetic Macrocycle

To evaluate clinical activity, response rates and duration of response based on IWG (2014) criteria or other appropriate criteria are provided with a case-by-case description of all patients who exhibit CR, PR or SD. A descriptive analysis of other evidence of anti-liquid cancer cell activity or other clinical benefit is provided based on clinical, radiographic or other appropriate assessment of efficacy or clinical anti-liquid cancer cell activity. Analysis of clinical activity is conducted on two patient populations: (1) the subset of patients who receive at least one cycle of therapy and have at least one post-baseline disease assessment (the efficacy-evaluable population) and (2) a larger group of patients that includes the efficacy-evaluable population as well as patients who exhibit objective disease progression or experience a DLT and/or unacceptable toxicity prior to the end of Cycle 1.

Imaging scans, physical examination, and/or laboratory-based assays (e.g., prostate specific antigen) for patients with relevant disease indications are obtained at baseline (e.g., within 28 or 21 days of Cycle 1 Day 1), including, for example, a baseline PET-FDG and possibly FLT-PET scan(s) and for objective anti-liquid cancer cell activity as oulined below. The same type of imaging, physical examination, or laboratory-based assay procedure is used for each assessment for a patient. IWG (2014) criteria are used to assess liquid cancer response and duration of response. Scheduled scans (and/or other laboratory-based assay) are interpreted prior to the start of the next treatment cycle. If the criteria for a CR or PR are met, then the scan is repeated no earlier than within 4 weeks to confirm the response. A responding patient (CR, PR or SD) continues on study, with disease assessment after Cycle 2 and every other cycle thereafter in DR-A (e.g., Cycles 4 and 6) and after the last infusion in Cycle 3 and every third cycle thereafter in DR-B (e.g., Cycles 6 and 9), until disease progression, withdrawal of informed consent, or unacceptable toxicity.

Films or other records from imaging procedures, including those procedures performed at a regional or other facility outside of the primary institutions, are read and reviewed by the radiology staff at the corresponding primary study institution for the patient.

[¹⁸F]-fluorodeoxyglucose positron emission tomographic (FDG-PET) imaging is performed at baseline using using IWG (2014) criteria for patients with liquid lymphoma. FDG-PET imaging post-baseline is only performed in patients at the first occurrence of stable disease as an adjunct to determine anti-liquid cancer cell activity. PET/CT scans can substitute for contrast-enhanced CT scans provided the CT performed as part of a PET-CT is of similar diagnostic quality as a diagnostic CT with IV and oral contrast.

FLT-PET imaging is performed at baseline for patients with liquid cancer cells commonly showing sufficient uptake of FLT tracer, e.g., patients with liquid lymphoma and other liquid cancers.

-   -   DR-A assigned patients who demonstrate a standard uptake value         (SUV) of ≥5 at baseline will have a repeat FLT image one day         after their last infusion of study medication in Cycle 1, i.e.,         Day 16.     -   DR-B patients who demonstrate a standard uptake value (SUV) of         ≥5 at baseline will have a repeat FLT image one day after their         last infusion of study medication in Cycle 1, i.e., Day 12.

Example 8: Molecular Screening Prior to Day 1 of Cycle 1

Molecular screening encompasses the following prior to the first administration of peptide 1 (Day 1 of Cycle 1).

-   -   Collection of signed informed consent for molecular screening     -   Collection of an archived liquid cancer cell sample or a fresh         liquid cancer cell biopsy (unless a biopsy poses significant         clinical risk) for p53 testing         -   If confirmed to be p53 WT, the remainder of the tissue             sample from enrolled patients are used to test for PD             biomarkers     -   Confirmation of p53 WT status before administration of the first         dose of peptide 1 is mandatory for enrollment in:         -   Stage 1 of DEP for patients starting at Dose Level 4 and             higher         -   Stage 2 (if necessary) of DEP and EXP for all patients

At Dose Level 4 and higher in Stage 1 of the DEP (as well as for all patients enrolled in Stage 2 of the DEP), molecular screening in patients with unknown p53 status is done prior to initiating the clinical screening. If the p53 status is known to be WT, these patients can proceed to clinical screening and can be enrolled and receive peptide 1 before confirmation of p53 WT by the central laboratory.

In the EXP, patients must have completed molecular screening at the central laboratory prior to proceeding to enrolment. These patients can only be enrolled and receive peptide 1 after confirmation of p53 WT by the central laboratory.

Example 9: Management of Tumor Lysis Syndrome (TLS)

There is a potential for tumor lysis syndrome (TLS) in patients with liquid lymphoma, especially those with large liquid cancer cell burden, pre-dose elevated lactate dehydrogenase and leukocyte counts, renal dysfunction, or dehydration. TLS can be caused by treatment-induced abrupt cancer cell disintegration. It is usually observed shortly after initiating treatment. Patients at risk for TLS can receive liquid cancer cell lysis prophylaxis as part of standard of care according to local clinical protocols.

Laboratory TLS is defined as a 25% increase in the levels of serum uric acid, potassium, or phosphorus or a 25% decrease in calcium levels. Therefore, patients are monitored for these laboratory parameters 24 hours (Day 2) after infusion of peptide 1 on Day 1 in DR-A and DR-B during Cycle 1. Signs and symptoms of TLS are monitored until resolved.

Example 10: Multiplexed Cytotoxicity Assay

The objective of this study was to concurrently evaluate peptide 1-induced cytotoxicity, p21 up-regulation and caspase-3 activation in multiple liquid cancer cell cell lines characterized as having either wild-type or mutated p53 in order to determine whether the cytotoxicity is PD-mediated.

Cell lines from hematologic liquid cancer cells, characterized for p53 mutant/wild-type status, were seeded into 384-well plates and incubated in a humidified atmosphere of 5% CO₂ at 37° C. Peptide 1 was serially diluted and assayed over 10 concentrations, up to 30 μM, in a final assay concentration of 0.1% DMSO. Treatment was added 24 hours post cell seeding, when a time zero untreated cell plate was also generated to determine the number of doublings in the 72 hour assay period. About one hundred cells per well were evaluated, with each test condition run in duplicate wells. After a 72 hour incubation period, cells were fixed and stained with fluorescently labeled antibody and nuclear dye. In the same wells, nuclei were assessed for toxicity (dye uptake) by automated fluorescence microscopy; apoptosis was assayed by elevations in anti-active caspase-3 antibody; and cell cycle arrest was assayed by elevations in anti-p21 monoclonal antibody EA10. Characteristics of the 32 hematologic cell lines evaluated in this study are listed in Table 15.

TABLE 15 Hematologic Tumor Cell Lines Evaluated for Cytotoxicity and for Induction of Caspase and p21 following 72-Hour Incubations with peptide 1 Mutant p53 Wild-Type p53 Tumor Tumor Cell Line Origin Cell Line Origin J-RT3-T3-5 Leukemia MOLT-3 Leukemia MEG01 Leukemia CML-T1 Leukemia CCRFCEM Leukemia MV-4-11 Leukemia MHH-PREB -1 Leukemia BV-173 Leukemia K562 Leukemia NALM-6 Leukemia EM-2 Leukemia SR Lymphoma CEM-C1 Leukemia Daudi Lymphoma Thp1 Leukemia DOHH-2 Lymphoma Jurkat Leukemia CRO-AP2 Lymphoma HEL-92-1-7 Leukemia RPMI 6666 Lymphoma MOLT-16 Leukemia BC-1 Lymphoma DB Lymphoma L-428 Lymphoma Ramos (RA 1) Lymphoma HT Lymphoma ST486 Lymphoma Raji Lymphoma EB-3 Lymphoma RPMI 8226 Myeloma ARH-77 Myeloma U266B1 Myeloma

Cell proliferation was measured by relative cell count, which was expressed as percent of control. The activated caspase-3 marker labels cells from early to late stage apoptosis, and output is shown as a fold increase of apoptotic cells over vehicle background normalized to the relative cell count in each well (Emax); a ≥5-fold induction indicates significant induction of apoptosis. For example, peptide 1 was able to induce robust apoptotic responses in p53 wild-type hematopoietic cell lines (see FIG. 2).

Total p21 level indicates relative activity of p53, and output is shown as a fold induction of p21 concentrations over vehicle background normalized to the relative cell count in each well (Emax); a ≥2-fold increase or decrease in total p21 protein per cell is considered significant. For example, peptide 1 was able to show significant induction of p21 and yielded on-mechanism p21 pharmacodynamic responses in p53 wild-type hematopoietic cell lines (see FIG. 3).

For each cell line, an EC₅₀ estimated the concentration at which 50% of the cells were killed. Additional parameters that characterized the responses include IC₅₀ (peptide 1 concentration at 50% maximal possible response), GI₅₀ (concentration need to reduce growth by 50%), and activity area (the integrated area over the survival curve). For example, proliferation and survival of cell lines with wild-type p53 protein was sensitive to peptide 1, with IC₅₀ values ranging from 0.2 to 3.3 μM. Not all cell lines exhibited cytotoxicity at the peptide 1 concentrations tested. Those that did were most often characterized by significant inductions of both caspase-3 and p21. Results with BV-173 leukemia cells (Table 16) are representative of the typical responses with these cell lines.

TABLE 16 Apoptotic Cytotoxicity in Wild Type P53-Containing BV-173 Tumor Cells Relative Cell Caspase Fold p21 Fold Conc. Count (%) Induction Change (μM) Mean SD Mean SD Mean SD 9.55E−04 100.8 2.7 1.1 0.2 1.1 0.1 3.02E−03 99.9 4.5 1.1 0.3 1.0 0.1 9.53E−03 94.9 0.9 1.2 0.2 1.0 0.1 3.01E−02 84.8 2.8 1.4 0.2 1.0 0.1 9.52E−02 64.4 2.9 2.4 0.7 1.1 0.2 3.01E−01 31.8 2.3 4.1 1.0 1.3 0.1 9.51E−01 14.3 0.3 6.3 1.1 2.3 0.3 3.00E+00 15.0 8.3 3.7 2.0 3.3 0.9 9.49E+00 4.3 0.5 N/A N/A N/A N/A 3.00E+01 2.2 1.1 N/A N/A N/A N/A

Cytotoxicity in liquid cancer cell lines with mutant p53, when observed, was most often characterized by significant induction of p21 without significant caspase-3 induction. Results with HEL-92-1-7 leukemia cells (Table 17) are representative of the typical responses with these cell lines.

TABLE 17 Non-Apoptotic Cytotoxicity in Mutant P53-Containing HEL-92-1-7 Tumor Cells Relative Cell Caspase Fold p21 Fold Conc. Count (%) Induction Change (μM) Mean SD Mean SD Mean SD 9.55E−04 98.5 4.9 1.2 0.2 1.3 0.2 3.02E−03 96.8 6.3 1.2 0.1 1.5 0.1 9.53E−03 98.2 2.6 1.1 0.1 1.7 0.2 3.01E−02 89.8 1.7 1.0 0.1 2.2 0.2 9.52E−02 89.7 2.6 1.0 0.2 2.9 0.0 3.01E−01 77.8 2.3 1.1 0.1 3.2 0.1 9.51E−01 81.9 7.7 1.1 0.2 3.7 0.3 3.00E+00 79.2 4.3 1.1 0.1 3.9 0.4 9.49E+00 76.9 1.8 0.7 0.1 4.4 0.7 3.00E+01 67.9 5.1 0.6 0.1 4.2 0.6

Using an EC₅₀ cut-off of 1 μM for all hematologic liquid cancer cell lines, induction of apoptosis was found to have very good agreement with the p53 status of the cells (Table 18). Therefore, p53 status can be a sensitive biomarker for testing cytotoxicity of compounds, such as peptide 1.

TABLE 18 Sensitivity of Tumor Cells Containing Wild-Type and Mutant p53 to peptide 1-Induced Cytotoxicity EC₅₀ for Cytotoxicity Wild Type p53 Mutant p53 >1 μM 11 160 ≤1 μM 60 2 Peptide 1 selectively induced p53-mediated apoptotic cell death in liquid cancer cell lines (e.g. hematopoietic cancer cells) containing wild-type p53 protein. As shown in FIG. 4, all eleven p53 WT (6 lymphoma and 5 leukemia) hematologic cancer cell lines were highly sensitive to peptide 1 intervention as all lines exhibited EC₅₀ less than 0.6 μM. Taken together, these lines of evidence suggested effectiveness of peptide 1 against liquid tumor cell lines across multiple histological origins that retain the p53 WT status. In liquid cancer cell lines containing mutated p53, cytotoxicity was associated with cell cycle arrest without elevated apoptosis. These findings demonstrate the pharmacodynamic selectivity of peptide 1 towards liquid cancer cells with active wild-type p53.

Example 11: The Effect of Peptide 1 on Tumor Growth Inhibition

In another study, MV 4; 11 human leukemia xenograft model was used in mice to assess the ability of peptide 1 to inhibit tumor growth and improve overall survival in AML, a highly aggressive liquid tumor. In this study, 25 mg/kg dose of peptide 1 was administered in six bi-weekly doses and compared to the results of mice treated with only cyclophosphamide, the control group. Mice were monitored individually for an endpoint of morbidity due to progression of the leukemia. All ten mice that received the control exited the study between days 21 and 28, offering a sensitive assay for activity. As shown in FIG. 5, treatment with peptide 1 resulted in median overall survival of 40 days as compared to 22 days for untreated mice, an 81% increase for those receiving peptide 1. Peptide 1 can have an effect in liquid tumors with WT p53 and in AML.

Example 12: The Safety and Toxicology of Peptide 1

The 4-week multiple-dose GLP studies in rats and monkeys utilized twice-weekly IV dosing of peptide 1. The studies provided dose- and exposure-related assessments during both dosing and recovery periods, and results were utilized to define the maximum tolerated doses (MTD) and estimate the severely toxic dose for 10% (STD₁₀) of rats and the highest non-severely toxic dose (HNSTD) in monkeys. All gross and microscopic signs of intolerance (e.g., reduced organ weights, sporadic findings of multi-tissue hemorrhage and hepatic necrosis) and changes in serum chemistry parameters were considered as secondary to red blood cell (RBC), platelet and/or white blood cell (WBC) depletions or anorexia and dehydration in both species. Recovery assessments revealed regenerative and compensatory changes consistent with marrow cell survival and reversibility of all related hematologic and secondary toxicities.

The dose limiting toxicities (DLT) in both animal species appears to be related to the suppression of hematopoietic cells in the bone marrow, in particular cells of the megakaryocyte lineage, resulting in significant decreases in peripheral blood platelets that demonstrated recovery upon the cessation of dosing. For example, dose-dependent decreases in platelets with recovery were shown in a representative 4-week monkey GLP toxicity study using different dosings of peptide 1 (see FIG. 6).

The STD₁₀ in rats was determined at 10 mg/kg based on the mortality of one animal in a satellite group for hematology sampling during recovery. The HNSTD in monkeys was determined at 5 mg/kg, based on a complete lack of significant thrombocytopenia at this lowest dose level. However, almost all of the monkeys at the mid- and high-dose levels tolerated peptide 1 administration well; only one animal at each of these dose levels developed significant thrombocytopenia (<100,000×10⁶/ml).

Rats were more sensitive to the bone marrow and hematologic effects of peptide 1 than monkeys on the basis of exposures at maximally tolerated doses. Exposure at rat STD₁₀ (AUC_(0-∞)=562 μg·hr/mL at 10 mg/kg) was below that of HNSTD in monkeys (AUC_(0-∞)=813 μg·hr/mL at 5 mg/kg). These in vivo results correlated with those obtained from in vitro hemotoxicity assays via luminescence output (HALO). In these investigations, peptide 1 in general inhibited the induced proliferation of bone marrow precursor cells from rats to a greater extent than those from monkeys or humans. IC₅₀ values were ˜2- to 8-fold higher for rat cells than for monkey or human cells, with the largest difference noted for megakaryocyte colony forming cells, the platelet precursors. These results correlated with in vivo findings indicating that rats are more sensitive to the bone marrow and hematologic effects of peptide 1 than monkeys on the basis of dose and exposures at maximally tolerated doses. These results also suggested that, in terms of projecting potential bone marrow and hematological toxicity levels in humans, the monkey PK-PD data can be more clinically relevant than the rat data.

Peptide 1 was negative in genetic toxicology studies, including bacterial mutagenicity (Ames), chromosomal aberrations (human peripheral blood lymphocyte) and in vivo micronucleus (rat bone marrow) assays. Safety pharmacology studies were performed to assess the effects of peptide 1 on hERG potassium channels in vitro and on cardiac function in cynomolgus monkeys.

The above studies demonstrated that peptide 1 showed a favorable profile in preclinical GLP safety studies in rodents and monkeys. Genotoxicity, gastrointestinal toxicity, cardiotoxicity, and immunogenicity were not observed. Histopathology showed bone marrow hypocellularity consistent with mild to moderate myelosuppression.

Example 13: Pharmacokinetics of Peptide 1

In rats, peptide 1 generally showed linear, dose-proportional increases in C_(max) and AUC. In the 4-week rat GLP toxicity study, C_(max) of peptide 1 ranged from 49.9 to 186 μg/mL, AUC_(0-∞) ranged from 90.5 to 562 g·hr/mL, and clearance ranged from 19.2 to 28.3 mL/hr/kg.

In non-human primates, peptide 1 generally showed exposures that increased proportionally with dose, although an apparent plateau in exposure was observed at the high-dose group (20 mg/kg) in the 4-week monkey GLP toxicity study. In the study, C_(max) of Aileron peptide 1 ranged from 133 to 562 μg/mL, t_(1/2) ranged from 3.7 to 6.0 hrs, AUC_(0-∞) ranged from 813 to 1,600 μg·hr/mL, and clearance ranged from 6.5 to 13.8 mL/hr/kg.

No significant sex-based differences in PK parameters were observed in either rats or monkeys, and no accumulation was observed following repeated doses on a twice-weekly schedule in the GLP toxicity studies.

In vitro studies revealed that peptide 1 is not an inhibitor of any cytochrome P450 (CYP) isoforms tested. In vitro assays for CYP induction also did not indicate any significant treatment-related effects with peptide 1. Based on these findings, the potential of clinically relevant drug-drug interactions for concomitant medications that are cleared through CYP-mediated mechanisms can be low. 

1.-210. (canceled)
 211. A method of treating a liquid tumor in a human subject in need thereof, wherein the method comprises administering to the human subject a therapeutically effective amount of a p53 activator, wherein the p53 activator is a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, and wherein the p53 activator binds to a MDM2 or a MDMX protein.
 212. The method of claim 211, wherein the p53 activator binds to a MDM2 protein and disrupts an interaction between a p53 protein and the MDM2 protein.
 213. The method of claim 211, wherein the p53 activator binds to a MDMX protein and disrupts an interaction between a p53 protein and the MDMX protein.
 214. The method of claim 211, wherein the liquid tumor has no p53 deactivating mutation.
 215. The method of claim 214, wherein the liquid tumor expresses a wild type p53 protein.
 216. The method of claim 214, wherein the liquid tumor has a p53 gain of function mutation.
 217. The method of claim 214, further comprising determining that the liquid tumor lacks the p53 deactivating mutation prior to administering the p53 activator.
 218. The method of claim 211, wherein the administering is intravenous.
 219. The method of claim 211, wherein the administering is once every week.
 220. The method of claim 211, wherein the administering is once every 2 weeks.
 221. The method of claim 211, wherein the administering is once every 3 weeks.
 222. The method of claim 211, wherein the administering is two times a week.
 223. The method of claim 211, wherein the administering is three times a week.
 224. The method of claim 211, further comprising administering a therapeutically effective amount of at least one additional therapeutic agent to the human subject.
 225. The method of claim 211, wherein the treating causes essentially no adverse effects in a normal-hematopoietic tissue in the human subject.
 226. The method of claim 211, wherein the treating results in essentially no dose-limiting thrombocytopenia in the human subject.
 227. The method of claim 211, wherein the therapeutically effective amount is about 0.5 to about 10 mg per kilogram body weight of the human subject.
 228. The method of claim 211, wherein the liquid tumor is myelodysplastic syndrome, acute myeloid leukemia, multiple myeloma, neoplasm, or chronic myeloproliferative disorder.
 229. The method of claim 211, wherein (i) the liquid tumor is acute myeloid leukemia (AML), or (ii) the liquid tumor is not a HPV positive cancer; or (iii) the liquid tumor is peripheral T cell lymphoma; or (iv) the liquid tumor is a myelodysplastic syndrome (MDS); or (vi) the liquid tumor is chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, unspecified, anaplastic large cell lymphoma, classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte depleted or not depleted), or nodular lymphocyte-predominant Hodgkin lymphoma.
 230. A pharmaceutical composition comprising a p53 activator for treating a liquid tumor in a human subject in need thereof, wherein the p53 activator is a peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof, wherein the p53 activator binds to a MDM2 or a MDMX protein. 